A heteromeric TRP channel that functions as a WNT-activated G protein-coupled receptor

Domain: chemistry
Published: 2026-02-26
Editorial outcome: minor_revision

Abstract
The human genome contains approximately 800 G protein-coupled receptors (GPCRs), all characterized by a common 7-transmembrane domain architecture. Here, we show that PKD1, an 11-transmembrane protein with a noncanonical transient receptor potential (TRP) channel architecture, functions as a GPCR with unique biochemical properties. PKD1 acts as a WNT-activated receptor, directly coupling to heterotrimeric G\u03b1 subunits to inhibit cellular cAMP accumulation. While PKD1 contains both ligand-binding and G protein recruitment sites, PKD2, an associating TRP channel subunit, chaperones PKD1 to the plasma membrane to operate as a GPCR. This represents a striking departure from classical GPCR architecture and expands the functional repertoire of the TRP channel family. Given that mutations in either PKD1 or PKD2 are linked to autosomal dominant polycystic kidney disease, a multisystemic disorder marked by elevated cAMP levels, our results provide molecular insights into disease pathogenesis and highlight potential new therapeutic avenues for this debilitating and costly condition.

Introduction
Vertebrate genomes contain ~1,000 G protein-coupled receptors (GPCRs), forming the largest class of cell surface receptors mediating a plethora of cellular functions. All known GPCRs have a structural signature consisting of a 7-transmembrance (TM) domain flanked by an extracellular N-terminal segment and an intracellular C-terminal tail. Coupling of these receptors to a diverse group of heterotrimeric G proteins leads to the generation of second messengers, such as signaling lipids, Ca2+, and cAMP, that initiate a complex array of cellular responses1-3.

Autosomal dominant polycystic kidney disease (ADPKD), a multisystemic genetic disease affecting roughly 12.5 million people worldwide,4, 5 has been mechanistically linked to altered cAMP metabolism6-9. ADPKD is caused by hetero zygous germline mutations in PKD1 and PKD2 genes10-12. PKD1 is a large (~500 kDa) integral membrane protein with 11 membrane-spanning segments13, an extensive extracellular N-terminal fragment, and an intracellular C-terminal tail14. PKD2 (~130 kDa) spans the membrane 6 times and belongs to the transient receptor potential (TRP) ion channel superfamily12, 15. PKD1 and PKD2 assemble to form a heteromeric complex with a noncanonical “TRP” channel architecture16 for which the function has not been clearly defined. A unique and paradoxical feature of the PKD1/PKD2 complex is that the side chains of three positively-charged residues of PKD1 protrude into the ionic pore of PKD2 rendering it unlikely to conduct Ca2+ and other cations. Thus, while these subunits do assume a TRP-like pore architecture, their function as a cation-permeable channel complex is unsettled.

Evidence accumulated over 30 years has shown that cells derived from ADPKD patients have abnormally high cytosolic cAMP levels6-9 and an aberrant response to cAMP17 , which is critical for disease development. Deletion of phosphodiesterase 1 and 3 family members (Pde1a, Pde1c, and Pd3a) aggravate PKD18, and pharmacologic inhibition of PDE4 promotes cystogenesis in mouse Pkd1-null embryonic kidneys19. Conversely, pharmacologic and genetic manipulations to lower cAMP suppress cyst progression and improve renal function20-22. These studies culminated in the identification of Tolvaptan, which serves as the only FDA-approved agent for the treatment of ADPKD23-25. Tolvaptan is a potent antagonist of the vasopressin 2 receptor26, which is coupled to the heterotrimeric G\u03b1 subunit leading to cAMP production. Whilst the use of Tolvaptan is limited to a subset of ADPKD patients due to side effects, these studies collectively suggest that elevated cellular cAMP concentration is a driver of cystogenesis in ADPKD. However, as the molecular function(s) of PKD1 and PKD2 remain elusive, a major knowledge gap is the mechanisms by which PKD1 and/or PKD2 keep cytosolic levels of cAMP in check.

Fragmented evidence suggests that PKD1 may function as an atypical GPCR27. Indirect support for the function of PKD1 as a GPCR comes from the presence of a GPCR-autoproteolysis-inducing (GAIN) domain in its ectodomain, a defining feature of cell adhesion GPCRs28, 29. However, there is no evidence linking autoproteolysis to cAMP regulation. Independent biochemical studies show that several G\u03b1 subunits can directly bind the C-terminal tail of PKD130, 31, and a naturally occurring PKD1 mutation (L4132\u2206) within a predicted G protein-binding motif leads to severe cystic disease32. Finally, functional studies using various cell culture heterologous systems33-37, a mouse model38, or the Xenopus pronephric model system support the role of PKD1 in GPCR-mediated signaling31. However, direct evidence that PKD1 functions as a GPCR in response to a specific ligand in any system is lacking. Combined with the fact that PKD1 lacks the typical ‘7-TM’ signature architecture of classical GPCRs, these knowledge gaps have left the questions open of whether PKD1 is truly a GPCR and, if so, how this function is linked to cellular cAMP levels.

Results
Activation of PKD1 by WNT ligands induces G\u03b1-G\u03b2\u03b3 dissociation
We have shown previously that WNT-9B functions as the activating ligand of PKD1 in a signaling pathway determining kidney tubular diameter during renal development39. Disruption of this pathway results in the failure of developing tubules to achieve and maintain the proper tubular diameter leading to cystogenesis40, 41. Here, we sought to determine whether PKD1 mediates heterotrimeric G protein activation in response to WNT-9B. Activated GPCRs promote the exchange of GDP for GTP on G\u03b1 subunits followed by the dissociation of G\u03b1-GTP and free G\u03b2\u03b31. To assess PKD1-mediated G\u03b1-G\u03b2\u03b3 dissociation, we employed a set of bioluminescence resonance energy transfer (BRET)-based G protein activity sensors (G-CASE) covering all four major G protein families42. These sensors comprise G\u03b1, G\u03b2, and G\u03b3 on a single plasmid, with G\u03b1 subunits tagged with the luminescent donor, NanoLuciferase (Nluc), and G\u03b3 subunits N-terminally labeled with circularly permuted Venus (cpVenus173) to serve as the acceptor fluorophore (Fig. 1a). We conducted \u2206BRET time courses for six of the available G protein sensors in human embryonic kidney (HEK) 293T cells transiently co-transfected with the different biosensors and PKD1 (Fig. 1b-g). Kinetic analysis covered a time span of 10 minutes, 1 minute before and 9 minutes after stimulation with 1 \u00b5g/ml (30 nM) WNT-9B or vehicle control.

WNT-9B did not induce the dissociation of G\u03b1 or G\u03b1 (Fig. 1b and c). However, WNT-9B induced PKD1-mediated dissociation of G\u03b1 and G\u03b1 , with the largest response seen for G\u03b1 (Fig. 1d-h). The EC for WNT-9B was 405.6 ng/ml (11 nM) (Fig. 1i), which was consistent with the estimated EC of WNT-9B binding to extracellular fragments of PKD139 and channel activation43. The magnitude of PKD1-mediated G\u03b1 activation by WNT-9B was comparable to the magnitude of responses elicited by the activation of known GPCRs, such as the muscarinic acetylcholine receptor M1 (M1R) and calcium-sensing receptor (CaSR) using G-CASE sensors (Supplementary Fig. 1a and b). Cell surface expression of PKD1 was confirmed using a BRET-based saturation assay, human PKD1 C-terminally-tagged with Nluc (PKD1-Nluc) was titrated with increasing concentrations of a HaloTag-fused cell surface marker bearing the RAS CAAX motif44 (Supplementary Fig. 1c). Specificity of this interaction is indicated my saturable binding (Supplementary Fig. 1d). Live-cell imaging verified expression of PKD1-eGFP at the cell surface, as seen by co-localization with HaloTag-fused CAAX (Supplementary Fig. 1e).

Next, we determined if G protein selectivity differs in response to various WNT family members, as we have shown that in addition to WNT-9B, WNT-5A and WNT-3A bind the extracellular domain of PKD139. These experiments offered a unique opportunity to compare (a) canonical/\u03b2-catenin dependent (WNT-3A), (b) noncanonical/\u03b2-catenin independent (WNT-5A), and (c) mixed (both canonical and noncanonical, i.e., WNT-9B) pathway activating WNTs. Similarly to WNT-9B, neither WNT-5A nor WNT-3A stimulated coupling between PKD1 and G\u03b1 or G\u03b1 (Fig. 1b and c). PKD1-G\u03b1 selectivity in response to WNT specificity is demonstrated by the reduction in the rate of G\u03b1 , G\u03b1 , and G\u03b1 dissociation from G\u03b2\u03b3 induced by WNT-3A compared to other WNTs. WNT-3A induced slower dissociation of these subunits compared to WNT-5A and WNT-9B (Fig. 1d-f and h). Maximal dissociation of G\u03b1 was seen with all three WNT ligands (Fig. 1g and h). The slower G\u03b1 dissociation by WNT-3A compared to the other two WNTs raises the possibility that ligand binding specificity, perhaps by involving different subdomains in the extracellular domain of PKD1 and/or co-receptors, determines selectivity in heterotrimeric G protein coupling. The overall BRET response and relative PKD1-mediated dissociation rates of all six G\u03b1 subunits in response to three WNT ligands are shown in the heat map diagrams (Fig. 1h).

FZD6 has been shown to couple to G\u03b1 and G\u03b1 subunits in response to WNT activation45, thus, we sought to determine whether FZD6 and possibly other FZDs endogenously present in HEK293T cells could account for PKD1-induced G\u03b1 -CASE activation in response to WNT-9B. ZNRF3 is an E3 ubiquitin ligase that constitutively clears FZDs and LRP6 from the cell surface46. Co-expression of ZNRF3 and PKD1 did not affect the magnitude or kinetics of WNT-9B-induced G\u03b1 dissociation from G\u03b2\u03b3, indicating that endogenous FZDs could not account for the activation of the G\u03b1 -CASE sensor by PKD1 (Supplementary Fig. 1g). The functionality of ZNRF3 was confirmed by its effect on overexpressed FZD6-induced activation of G\u03b1 -CASE (Supplementary Fig. 1h). To provide additional evidence for the preference of PKD1 towards G\u03b1 we determined the effect of G protein signaling modulator 1 (GPSM1), which functions as a G\u03b1 -specific guanine nucleotide dissociation inhibitor47, on WNT-9B-induced dissociation of G\u03b1 via PKD1. Co-expression of GPSM1 suppressed PKD1-mediated G\u03b1 dissociation in response to WNT-9B (Supplementary Fig. 1i). Altogether, these data suggested that WNT ligands induce the dissociation of G\u03b1 and G\u03b1 , but not G\u03b1 or G\u03b1 subunits from G\u03b2\u03b3 via PKD1. These effects do not seem to involve endogenous FZD receptors and LRP6 co-receptors in transiently transfected HEK293T cells.

WNTs did not induce significant G\u03b1 dissociation for any of the six subtypes in mock (sensor only)-transfected cells in response to WNT ligands (\u22645% drop in normalized BRET, Supplementary Fig. 1f) 48 hours following transfection or 24 hours post-seeding onto 96-well plates. However, 72 hours after transfection or 48 hours post-seeding, cells showed higher activity (up to 20% drop in normalized BRET) in response to WNT-9B in the absence of transfected PKD1 (Fig. 1j), perhaps by allowing more time to recover endogenous PKD1 from trypsinization after replating. We took advantage of this high signal-to-noise BRET ratio under these conditions to test whether endogenous PKD1 contributed to the WNT-induced activation of G\u03b1 . We knocked out human PKD1 using a combination of 3 human PKD1-specific sgRNAs and tested for WNT-9B-induced reduction of the G\u03b1 -CASE sensor activity 72 hours following transfections (Fig. 1j). Knockout efficiency was quantitatively determined (95%) on PKD1-Nluc 72 hours following transfection (Fig. 1k). Knockout of endogenous PKD1 led to a reduction in normalized BRET compared to sensor-only-transfected cells in response to WNT-9B. To confirm that this effect was PKD1-specific, we added back mouse PKD1, which is resistant to knockout by any of the 3 sgRNAs used to inactivate human PKD1. Mouse PKD1 rescued the effect of human PKD1 knockout (Fig. 1j, red versus purple). These data reveal an essential role of endogenous PKD1 in the WNT-9B-induced activation of G\u03b1 .

To further confirm the specificity of our results and obtain mechanistic insights of how PKD1 functions as a WNT-activated GPCR, we tested the effects of several PKD1 mutations (Fig. 2a) on G\u03b1 dissociation from G\u03b2\u03b3 in response to WNT-9B (Fig. 2b-d). S99I is a pathogenic mutant that compromises PKD1 cell surface expression39, and thus, it should reduce receptor function. Indeed, expression of PKD1-S99I failed to induce G\u03b1-G\u03b2\u03b3 dissociation. Similar results were obtained with W139C, also a pathogenic mutation located within the WNT-9B binding site. A similar effect was seen by the L4132\u2206 mutation, which contains a pathogenic in-frame deletion of L4132 localized within a predicted G protein-binding domain in the C-terminal tail of PKD132. Interestingly, the T3409V mutation, which renders PKD1 unable to undergo GAIN-mediated autoproteolysis29, led to an approximately 50% reduction in heterotrimeric G protein signaling (G\u03b1 -CASE, Fig. 2d). This result is consistent with the hypomorphic function of this mutation in mice29. Finally, the longest known pathogenic mutation, S4213X, truncates PKD1 by deleting the majority of its cytosolic C-terminal tail including the coiled-coil domain essential for interaction with PKD2, and resulted in complete loss of receptor function. Expression levels of mutants in transiently transfected HEK293T cells are shown in Supplementary Fig. 2. Altogether, these data agree with in vivo effects, supporting a biological role of PKD1 as a WNT-activated GPCR.

Next, we wanted to assess the mechanism of G protein coupling selectivity for PKD1 and compare this with the mechanism of canonical GPCRs. In the conventional mechanism of G protein activation, the distal portion of the G\u03b1 C-terminal alpha helix engages the TM domains of the receptor during coupling (Fig. 3a and c)48, 49. We designed chimeras for G\u03b1 -CASE and G\u03b1 -CASE, in which the 16 final C-terminal amino acids were exchanged with that of the other (Fig. 3b and d) and conducted BRET assays as described previously to determine whether the G\u03b1 C-terminus is a key component of coupling selectivity for PKD1 in response to WNT-9B (Fig. 3e). As expected, the exchange of the G\u03b1 C-terminus for that of G\u03b1 (G\u03b1 ) led to a reduction in the maximum response compared to the wild-type G\u03b1 . Interestingly, the G\u03b1 to G\u03b1 C-terminal swap (G\u03b1 ) only resulted in a partial gain of function. A range of G protein sensor concentrations were tested to account for possible expression issues, yet the maximum response was still seen using the same concentration as the wildtype sensor (Supplementary Fig. 3a). We also confirmed these results were PKD1-specific, as sensor-only-transfected cells did not produce any response upon WNT stimulation (Supplementary Fig. 3b and c). In contrast to PKD1, G\u03b1-coupled CaSR and G\u03b1 -coupled beta-2 adrenergic receptor (\u03b2 AR) each showed a complete exchange in coupling selectivity correlating with the swap in G\u03b1 C-terminal motif (Fig. 3f and g), validating the functionality of the chimeras. Compared to the CaSR and \u03b2 AR, our results for PKD1 indicate that the G\u03b1 C-terminus contributes only partially to selective coupling and suggests additional/multiple interactions between PKD1 and the G protein may dictate selectivity. Thus, the mechanisms dictating G protein coupling selectivity of PKD1 is likely different from traditional 7-TM containing GPCRs.

WNT-9B induces the recruitment of G\u03b1 subunits to PKD1 and G\u03b1-GDP to G\u03b1-GTP exchange
A hallmark of heterotrimeric G protein activation downstream of a GPCR is the exchange of GDP for GTP on G\u03b1 subunits following the recruitment of G\u03b1 subunits to the receptor1. Thus, we sought to determine whether PKD1 and G\u03b1 and G\u03b1 subunits are recruited to PKD1 in response to WNT-9B activation. In this series of experiments, we used PKD1-Nluc and YFP-tagged G\u03b1 and G\u03b1 subunits (Fig. 4a). Saturation experiments revealed saturable and thus specific interactions between PKD1 and G\u03b1 or G\u03b1 subunits (Fig. 4b-e), and in response to WNT-9B, these subunits are recruited to PKD1 in a time-dependent manner (Fig. 4f-i). To determine whether WNT-9B induces a PKD1-dependent G\u03b1-GDP to G\u03b1-GTP exchange, we employed a collection of biosensors (ONE-GO sensors) to measure G protein activation50 (Fig. 4a). WNT-9B induced GDP to GTP exchange on G\u03b1 and G\u03b1 subunits (Fig. 4j-m), but not G\u03b1 or G\u03b1 (Supplementary Fig. 4a and b). Altogether, these results are in complete agreement with results obtained using G-CASE biosensors, further supporting the hypothesis that PKD1 is directly coupled to heterotrimeric G protein signaling for a selective subset of G\u03b1 subunits.

PKD2 enables PKD1 to function as a GPCR
PKD2 is endogenously expressed at detectable levels in HEK293T host cells (Supplementary Fig. 5a), which could influence the functional expression of PKD1 as a GPCR. Thus, we inactivated the endogenous PKD2 gene via CRISPR/Cas9-mediated genome editing in these cells using a doxycycline-inducible Cas9 vector (Supplementary Fig. 5a, lane 4). The efficiency of the PKD2-specific CRISPR construct was confirmed on transfected human PKD2 (Supplementary Fig. 5b, lane 4). Inactivation of human PKD2 led to the loss of PKD1 receptor function, indicating that endogenous PKD2 was essential for the receptor function of PKD1 in HEK293T cells (Fig. 5a and Supplementary Fig. 5c and 4, gray versus red). The functional dependence of PKD1 on endogenous PKD2 was also tested in a previously characterized clone of mouse inner medullary collecting duct (mIMCD-3) cells (Fig. 5b, gray versus red), in which endogenous Pkd2 has been permanently inactivated51. Therefore, the requirement of endogenous PKD2 for the functional expression of PKD1 as a GPCR is demonstrated in two different cell lines.

To gain insight into the mechanism by which PKD2 supported the receptor function of PKD1, we used the following PKD2 variants: D511V (or D509V in mouse PKD2), E442G (mouse PKD2), W201A (or W199A in mouse PKD2), and F604P (or F602P in the mouse). The D509V mutation compromises PKD2 channel activity and impairs maturation and targeting of PKD1 to the plasma membrane52. W199A reduces channel activity of PKD253, whereas F602P increases PKD2 channel activity54, 55. Finally, E442G does not affect channel function but impairs PKD2 trafficking to the primary cilium56. Homozygous Pkd2lrm4 (E442G) mice show a strong PKD phenotype, indistinguishable from that produced by complete Pkd1 or Pkd2 deletion57. Adding back PKD2-E442G or PKD2-D509V in PKD2-null cells failed to restore PKD1 activity (Fig. 5a, blue and green, respectively). Co-expression of PKD1-eGFP with wild-type untagged PKD2 or PKD2 C-terminally fused to HaloTag led to robust expression of both proteins at the plasma membrane (Fig. 5c and d). As expected, PKD2-D509V did not drive expression of PKD1-eGFP to the plasma membrane (Fig. 5c, D509V panels). Interestingly, PKD2-E442G also failed to target PKD1-eGFP to the plasma membrane (Fig. 5c, E442G panels). PKD1-Nluc co-localization with HaloTag-CAAX determined by BRET quantifiably support live-cell imaging data (Fig. 5e). Notably, overexpression of wild-type PKD2 increased overall levels of PKD1 (Fig. 5e and Supplementary Fig. 2), possibly via protein-protein-mediated stabilization, as has been shown using C-terminal tails of PKD1 and PKD258 . PKD2-E442G showed reduced stabilization of PKD1 compared to wild type (Fig. 5e), implying that E442G may affect PKD1/PKD2 assembly. The E442G mutation is located in the TOP domain of PKD2, which forms a tetrameric structure with 4-fold symmetry in homomeric PKD2 complexes59 and a similar structure but with a 15\u00b0 deviation from 4-fold symmetry in the heteromeric PKD1/PKD2 complex16. Since E442G did not impede channel function56, we reasoned that it should not affect self-assembly but rather assembly with PKD1. As expected, BRET experiments showed highly-efficient energy transfer between wild-type PKD1-Nluc and PKD2-HaloTag (Fig. 5f). However, BRET was severely reduced by the E442G mutation (PKD2-E442G-HaloTag), suggesting that E442G interferes with PKD1/PKD2 complex assembly and thus fails to target PKD1 to the plasma membrane. Expression of human PKD2-W201A or human PKD2-F604P in HEK293T cells did not affect PKD1 expression at the plasma membrane (Fig. 5c, W201A and F604P panels). In agreement with this finding, expression of mouse PKD2-W199A or mouse PKD2-F602P in Pkd2-null HEK293T cells and human PKD2-W201A or human PKD2-F604P in Pkd2-null mIMCD-3 cells did not impair G\u03b1 dissociation from G\u03b2\u03b3 in response to WNT-9B (Fig. 5a and b). Furthermore, treatment of wild-type mIMCD-3 cells with La3+ which blocks cation permeation of PKD1/PKD239, PKD2, or even structurally related PKD2-L160 did not have a significant effect on the GPCR function of PKD1 (Fig. 5b). Collectively, these data suggest that PKD2 enables PKD1 to function as a GPCR by chaperoning it to the plasma membrane, independently of its channel function (Fig. 5g). They further implicate the loss of PKD1 GPCR function as an alternative mechanism underlying the cystogenesis observed in Pkd2lrm4 (E442G) mice57.

PKD1-mediated G\u03b1 activation inhibits cAMP accumulation
G\u03b1 signaling leads to inhibition of cAMP accumulation (Fig. 6a). Therefore, we tested whether overexpression of PKD1 can inhibit basal or forskolin-induced cAMP accumulation in response to WNTs. We employed GloSensor technology, a cAMP assay to monitor changes in cytosolic cAMP levels in PKD1-transfected cells using two complementary strategies: (i) cells were treated with a concentration of forskolin close to its EC (10-6M) (Fig. 6b and c) and soluble WNTs were added when cAMP had reached its half-maximal concentration (Fig. 6c). In strategy (ii), endpoint analysis was used whereby PKD1-transfected cells were pretreated overnight with pertussis toxin (PTX, 100 ng/ml) to block G\u03b1 signaling, then treated with forskolin in the presence or absence of soluble WNTs for 10 minutes and cAMP levels determined (Fig. 6d). Kinetic analysis showed that WNTs suppressed the dynamic accumulation of cAMP via PKD1 (Fig. 6c). WNT-3A had the lowest effect (Fig. 6c), which was consistent with the fact that it is most efficiently coupled to G\u03b1 (Fig. 1g and h), which shows lower endogenous expression than G\u03b1 in HEK293T cells (Supplementary Fig. 6a). G\u03b1 is practically undetectable in HEK293T cells (Supplementary Fig. 6a). Similar results were obtained using the endpoint analysis, whereby PTX suppressed WNT-induced inhibition of cAMP accumulation (Fig. 6d). We next asked whether WNT ligands can lower basal (resting) levels of cytosolic cAMP in the presence of PKD1. WNT-9B induced a time-dependent reduction in cytosolic cAMP concentration in cells transfected with PKD1 (Fig. 6e, left trace). A similar but smaller effect was seen with WNT-5A or WNT-3A (Fig. 6e, center and right traces, respectively). The PKD1-mediated reduction in basal cAMP levels in response to WNT-9B was approximately half of the response elicited by the well-characterized muscarinic acetylcholine receptor M2 (M2R) following activation with carbachol in HEK293T cells (Supplementary Fig. 6b). Expression of pathogenic PKD1 mutants showed markedly suppressed inhibition of steady-state cAMP levels compared to wild-type PKD1 (Fig. 6f). To determine whether PKD2 had an essential role in WNT-induced inhibition of cAMP concentration via PKD1, we used our endpoint analysis for cAMP in cells lacking endogenous PKD2 and cells in which wild-type PKD2 was added back. These experiments support the role of PKD2 in inhibiting forskolin-induced cAMP accumulation in response to WNT-9B (Fig. 6g). We confirmed these results in wild-type and Pkd2-null mIMCD-3 cells using the end-point analysis. WNT-9B produced a significant reduction in forskolin-induced cAMP levels in wild-type, but not in Pkd2-null cells (Fig. 6h). Interestingly, deletion of Pkd2 did not affect resting cAMP levels. In summary, these data suggest that G\u03b1 signaling mediated by WNT-induced activation of PKD1 is sufficient to reduce steady-state and forskolin-induced cAMP accumulation and this regulation is compromised in Pkd2-null cells (Fig. 6i).

GRK6 promotes the internalization of PKD1 and suppresses PKD1-mediated signaling
Typically, GPCR signaling is regulated through the process of desensitization, whereby G protein-coupled receptor kinases (GRKs) are recruited to the activated receptor to phosphorylate specific sites, “priming” the receptor for recruitment of beta-arrestins (ARRBs), which signal receptor internalization and degradation or recycling. Alternatively, GRK recruitment can initiate ARRB-dependent signaling that is independent of G\u03b1/G\u03b2\u03b3 signaling. In either case, some intrinsic affinity of the receptor for GRKs is needed to initiate desensitization and/or ARRB-based signaling. Based on this, we tested whether PKD1 is localized proximally to GRK2, GRK3, GRK5, or GRK6. BRET experiments using PKD1-Nluc and the different GRK subtypes fused to HaloTag revealed saturable and specific recruitment of GRK2, GRK5, and GRK6, but not GRK3 to PKD1-Nluc (Fig. 7a-d). Interestingly, the maximum BRET between PKD1 and GRK6 was increased upon overexpression of PKD2, but to a lesser degree by overexpression of PKD2-\u2206C which lacks the C-terminal tail of PKD2 (Fig. 7d) and fails to target PKD1-eGFP to the plasma membrane (Fig. 7f). The opposite effect was seen with GRK2 (Fig. 7c). Since GRK2 is expressed predominantly in the cytoplasm, while GRK6 is anchored to the plasma membrane via palmitoylation modifications in its C-terminus61, these data suggest that PKD2 targets the PKD1/PKD2 complex to the plasma membrane where the complex is then in proximity to GRK6. This interpretation is consistent with the reduction in BRET between PKD1-Nluc and GRK2-HaloTag in the presence of PKD2 (Fig. 7c). The PKD2-\u2206C construct fails to facilitate co-localization of PKD1 with GRK6 (Fig. 7e, lower panels) because it does not target the complex to the cell surface. To determine whether GRK6 functionally affects PKD1 coupling to G\u03b1 , we tested whether it promotes internalization and negatively affects WNT-induced PKD1 signaling. Overexpression of GRK6 did not affect overall receptor levels (Fig. 7g, right bar graph) but did result in the reduction of PKD1 from the cell surface (Fig. 7g, left bar graph) and WNT-induced PKD1-mediated G\u03b1 dissociation from G\u03b2\u03b3 (Fig. 7h, DMSO). Inhibition of GRK6 activity using two different GRK6 inhibitors, GRK6-IN-1 or GRK6-IN-2, for 1 hour prior to WNT-9B activation partially reverted PKD1/PKD2-mediated G\u03b1 coupling in the presence of GRK6, indicating that the kinase activity of GRK6 was essential for its effect on PKD1/PKD2 function (Fig. 7h). Given the short-term effect of GRK6 inhibition (1 hour due to the toxicity of the inhibitors) on PKD1 internalization, we consider the partial reversal on PKD1/PKD2 function significant.

ARRB2 functions downstream of GRK6 in mediating internalization of the PKD1/PKD2 complex
PKD1 has been shown to interact with ARRB2 via its Polycystin-1, lipoxygenase, and \u03b1-toxin (PLAT) domain located in the intracellular loop between the first and seconf TM domains62. BRET saturation assays confirmed specific binding of HaloTag-fused ARRB2, but not ARRB1, to C-terminally-tagged PKD1-Nluc (Fig. 8a and b). Kinetic analysis revealed time-dependent recruitment of ARRB2-HaloTag to PKD1-Nluc in response to WNT-9B, which was unaffected by overexpression of PKD2 (Fig. 8c). However, time-dependent recruitment of ARRB2-HaloTag to Nluc-tagged PKD2 (PKD2-Nluc) was found to be PKD1-dependent (Fig. 8c), suggesting that recruitment of ARRB2 to PKD2 is indirect via PKD1 and that ARRB2 is recruited to the PKD1/PKD2 complex. WNT-9B-induced recruitment of ARRB2-HaloTag to PKD1-Nluc was reduced when cells were first incubated with GRK6 inhibitors, supporting the role of endogenous of GRK6 in the recruitment of ARRB2 to the PKD1/PKD2 complex (Fig. 8d).

To provide further evidence that the PKD1/PKD2 complex rather than PKD1 alone is necessary for GPCR function at the plasma membrane, we determined the kinetics for WNT-9B-induced internalization of PKD1-Nluc and PKD2-Nluc from the plasma membrane and for accumulation of these proteins in early endosomes. We reasoned that if PKD2 has an initial chaperone function to deliver PKD1 to the cell surface and then to dissociate from PKD1 after successful delivery, PKD2 would not show the same time-dependent internalization as PKD1 in response to a ligand. Fig. 8e-g shows that this is not the case. Steady-state BRET measurements and super-resolution imaging confirmed proximity and co-localization of PKD1-Nluc or PKD2-Nluc to HaloTag-Rab5 and HaloTag-FYVE1 (Fig. 8e and Supplementary Fig. 7) which marks the early endosome. WNT-9B stimulation in BRET time-course assays revealed similar kinetic profiles for PKD1-Nluc and PKD2-Nluc for both internalization and accumulation in the early endosome (Fig. 8f). The internalization of PKD1 in response to WNT-9B was inhibited by barbadin, an ARRB inhibitor (Fig. 8g). These results indicate that PKD2 remains associated with PKD1 during ligand activation and both proteins move together to the early endosome following activation (Fig. 8h).

Methods
Plasmids
Human wild-type PKD1-HA and human PKD1 T3049V were provided by Feng Qian. Human PKD1 L4132\u2206-HA and human PKD1 S4213X-HA were provided by Stephen Parnell32. Mouse PKD1-eGFP was provided by Chris Ward (University of Kansas Medical Center). Mouse Flag-PKD1 was provided by J. Yang (Columbia University). Human PKD1 gRNAs (1-3) and Cas9 were purchased from Addgene (cat #76841, #76842, #76843, and #52962, respectively). To generate human PKD1-Nluc, Nluc was cloned out of the pNLF1-C [CMV/Hygro] vector (Promega) and into PKD1-HA before the HA tag. Mouse PKD2 was made by PCR amplification of mouse cDNA derived from a mouse embryonic kidney cDNA library and cloned into pcDNA3. Human PKD1 W139C-HA39, human PKD1 S99I-HA 39, mouse PKD2 E442G, mouse PKD2 D509V, mouse PKD2 W199A, mouse PKD2 F602P, human PKD2 W201A, and human PKD2 F604P were generated using the QuikChange mutagenesis kit (Agilent Technologies). Human PKD1 \u2206C was generated by cytoplasmic C-terminal region deletion using pCS2+MT-6xMyc-hPKD2 (Addgene #21370). Mouse PKD2-HaloTag was generated by cloning the mouse PKD2 into the pHTC HaloTag CMV-neo vector (Promega), and mouse PKD2 E442G-HaloTag was generated by mutagenesis. Mouse PKD1-Nluc was generated by cloning the mouse PKD2 in the pNLF1-C [CMV/Hygro] vector (Promega). eTLCV-hPKD2 was generated by cloning the guide RNA for human PKD2 into a modified version (eTLCV2) of the TLCV2 vector backbone (Addgene #87360). Gs-CASE (Addgene #168124), G13-CASE (Addgene #168127), Gq-CASE (Addgene #168125), Gi1-CASE (Addgene #168120), Gi2-CASE (Addgene #168121), and Gi3-CASE (Addgene #168122) were gifts from Gunnar Schulte. The Gi3-s and Gs-i3 chimeras were made in the Gi3-CASE and Gs-CASE as follows: For the Gi3-s chimera, the final 16 C-terminal amino acids of human Gi3 were replaced with those of human Gs (CRDIIQRMHLRQYELL). For the Gs-i3 chimera, the final 16 amino acids of human Gs were replaced with those of human Gi3 (VTDVIIKNNLKECGLY). Galpha-s ONE-GO (Addgene #189731), Galpha-13 ONE-GO (Addgene #189740), Galpha-q ONE-GO (Addgene #189738), Galpha-i1 ONE-GO (Addgene #189735), Galpha-i2 ONE-GO (Addgene #189736), and Galpha-i3 ONE-GO (Addgene #189737) were gifts from Mikel Garcia-Marcos. Galpha-q-YFP, Galpha-i1-YFP, Galpha-i2-YFP, and Galpha-i3-YFP were generated by cloning out the IRES and Nluc components from the ONE-GO biosensors. Mouse GRK2-HaloTag, mouse GRK3-HaloTag, GRK5-HaloTag, human GRK6-HaloTag, ARRB1-HaloTag, ARRB2-HaloTag, HaloTag-CAAX. HaloTag-Rab5, and HaloTag-FYVE were obtained from Mohiuddin Ahmad. Human GRK6 and \u03b22AR were obtained from Augen Pioszak. M1R-HA and M2R were generated using RT-PCR from a human fetal kidney cDNA library. CaSR was obtained from Wenhan Chang (UCSF). Mouse FZD6 in SPORT6 was obtained from Horizon Discovery Collection of cDNA & ORF clones and verified by Sanger sequencing. ZNRF3 was obtained from Dr. Cong (Novartis). Human GPSM1 was purchased from Sino Biological (HG19737-UT). The pGloSensor-22F cAMP Plasmid was purchased from Promega.

Recombinant proteins and drugs
Recombinant human WNT-3A (5036-WN), human/mouse WNT-5A (645-WN), and mouse WNT-9B (3669-WN) were purchased from R&D Systems/Biotechne. WNTs were dissolved to 100 \u00b5g/ml in filter-sterilized in PBS containing 0.1% bovine serum albumin (BSA) and stored at -20\u00b0C in low-protein binding microcentrifuge tubes (Eppendorf). For stimulation experiments, WNTs were diluted in Opti-MEM I Reduced Serum Medium, no phenol red (Gibco) containing 0.1% BSA. The following drugs were stored at -20\u00b0C as stock solutions and diluted in PBS or assay medium on the day of experiments: R568, Spermine, Carbachol, Isoproterenol, Doxycycline, Forskolin (Thermofisher, J63292.MA), Pertussis Toxin (Thermofisher, PHZ1174), GRK6-IN-1 (MedChemExpress, HY-142812), GRK6-IN-2 (MedChemExpress, HY-142817), and Barbadin (MedChemExpress, HY-119706). Lanthanum was prepared from powder the day of the experiment (Sigma-Aldrich, 262072).

Cell lines
HEK293T cells (ATCC, CRL-3216) were maintained in DMEM (Corning) supplemented with 10% FBS (GeminiBio). Wild type and Pkd2-null mouse inner medullary collecting duct (mIMCD-3) cells were obtained from Dr. Steve Kleene51 and maintained in DMEM:F12 (Gibco) supplemented with 10% FBS. All cell lines were cultured in a humidified incubator at 37\u00b0C and 5% CO2. Absence of mycoplasma contamination was routinely confirmed by PCR.

Transient transfections
On the day of transfection, cells were trypsinized and diluted in cell culture medium to a final density of 2 \u00d7 105 cells/ml. Subsequently, 2 ml of cell suspension (400,000 cells) was plated in each well in a sterile six-well plate and incubated for 4-6 hours at 37\u00b0C, 5% CO2. HEK293T cells were transiently transfected using the calcium phosphate precipitation method. mIMCD-3 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendations. For all experiments, cells were transiently transfected with up to 4 \u00b5g total DNA per well in a 6-well plate. Transfected cells were then incubated for 24 hours at 37\u00b0C, 5% CO2. Plasmid amounts and combination used for transient transfections are detailed in Supplementary Table 2.

Bioluminescence resonance energy transfer assays
24 hours post-transfection, cells were trypsinized and resuspended in cell culture medium. Following centrifugation, cells were resuspended in assay medium (Opti-MEM I Reduced Serum Medium, no phenol red, supplemented with 4% FBS) at a final density of 2 \u00d7 105 cells/ml, and 100 \u00b5l dispensed per well of a sterile white 96-well microplate (Greiner Bio-One) and incubated for 24 hours at 37\u00b0C, 5 % CO2. For BRET assays using Nluc- and HaloTag-fused proteins, half of the cells were treated with 100 nM HaloTag NanoBRET 618 Ligand (Promega) and the other half with 0.1% DMSO (no-ligand control), and 100 \u00b5l of both cell suspensions dispensed into separate wells. 24 hours post seeding, 25 \u00b5l of furimazine (Promega) was added to each well (final dilution 1:1,000) and incubated for 5 minutes at room temperature. All measurements were performed using a Synergy Neo2 hybrid multi-mode microplate reader (BioTek) equipped with filters to simultaneously measure the donor and acceptor emissions (450/50 and 610LP for BRET experiments with Nluc- and HaloTag-fused proteins, or 460/540 for experiments with G-CASE sensors, ONE-GO sensors, or G\u03b1-YFP), and using an integration time of 200 ms. For stimulation experiments, baseline measurements were acquired for five minutes before manual addition of 25 \u00b5l of compound or vehicle control (assay medium) to the final concentration indicated in individual figure panels and/or legends. Signal was then measured for an additional 10-20 minutes. Assays done using BRET-based G protein biosensors were conducted at 27\u00b0C. Assays to measure Nluc- and HaloTag-fused protein interactions were conducted at room temperature. BRET data were collected using BioTek Gen5 software for detection (Agilent).

Glosensor cAMP assays
Approximately 400,000 HEK293T cells were seeded on each well of 6-well plates and transfected with plasmids 18 hours later. Cell medium was replaced 6 hours after transfections and cells incubated 24 hours at 37\u00b0C with 5% CO2. For experiments in which the PKD2 CRISPR KO was transfected, medium was supplemented with doxycycline (2 \u00b5g/ml). Subsequently, cells were rinsed with PBS, trypsinized, and resuspended in 2 ml of cell culture medium. Following centrifugation, cells were resuspended in fresh cell culture medium at a final density of 2 \u00d7 105 cells/ml, and 100 \u00b5l dispensed per well of a white 96-well microplate and incubated for 20 hours at 37\u00b0C, 5 % CO2. For end-point analyses to determine PTX-sensitivity, replacement medium was supplemented with 0.1 \u00b5g/ml PTX. The following day, medium was replaced with 100 \u00b5l of equilibration medium (88% CO2-independent medium, 10% FBS, 2% GloSensor cAMP Reagent stock solution in HEPES buffer) per well. Cells were then incubated for 2 hours at room temperature. For dose-response experiments to assess forskolin-induced cAMP accumulation detection using the pGloSensor-22F cAMP biosensor, a pre-read measurement was taken followed by the addition of varying concentrations of forskolin. Luminescence was measured after 10 minutes, and this value was divided by the pre-read measurement to determine fold response. For end-point analysis, a pre-read measurement was taken before compound addition. WNT ligands (1 \u00b5g/ml) or vehicle were then manually added to wells using a multichannel pipet and incubated for 5 minutes. Subsequently, forskolin (1 \u00b5M) was then manually added to all wells, and luminescence was measured 25 minutes later. For kinetic analysis, the plate was pre-equilibrated to the steady-state operating temperature of the microplate reader before compound addition by performing a pre-read kinetic measurement for 10 minutes to monitor the basal level of luminescence. For experiments to determine the inhibitory effect of WNTs on FSK-induced cAMP, following the pre-read kinetic measurement, 1 \u00b5M forskolin was added manually to each well using a multichannel pipet. Measurements were continued for 10 minutes, followed by manual addition of WNT (1 \u00b5g/ml) or vehicle control, and the plate was read for an additional 10 minutes. For experiments to measure the agonist effect on basal cAMP levels, WNT ligands (1 \u00b5g/ml) or vehicle were manually added following the pre-read kinetic measurement and recordings continued for 10 minutes.

Live-cell imaging
HEK293T cells were transfected in 8-well chambered cover glasses (Cellvis) with expression plasmids. After a 24-hour incubation period post-transfection, cells were labeled with 200 nM JFX 554 HaloTag Ligand (Promega) for 30 min and cultured in phenol red-free Opti-MEM. Live-cell imaging was done using an oil immersion 1.48 NA 100X objective lens in a Nikon CSU-W1 SoRa Spinning Disk confocal microscope. During image acquisition SoRa 2.8x magnification was used with laser power set at 85% 200 ms for 488 channel, and 10% 100 ms for the 561 channel, all with a step size of 0.1 \u00b5m. NIS Elements Software was used to process the images with the LUT set at 100-200 for the 488 channel.

Immunoprecipitations
In experiments where immunoprecipitations were done in lysates, cells were lysed in 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, and 10% sucrose with protease inhibitor cocktail (Roche Applied Science) at 4\u00b0C for 30 min and lysates were collected by centrifugation (18,000 \u00d7 g, 20 min). Lysates (500 \u00b5l) were incubated at 4\u00b0C for overnight with indicated antibodies in figures coupled to Sepharose beads. Beads were pelleted by centrifugation, and pellets were washed in lysis buffer three times for 20 min with rotation at 4\u00b0C. Immunoprecipitated proteins immunoblotted with indicated antibodies in figures.

qRT-PCR
RNA was isolated from HEK293T cells using TRIzol reagent (Thermofisher) and further purified using Qiagen RNA isolation kit (Qiagen). cDNA was prepared from 500 ng RNA using Maxima First Strand cDNA Synthesis Kit (Thermofisher). qRT-PCR assays were performed using EvaGreen 2X qPCR MasterMix (Bullseye) and primers corresponding to the appropriate cDNA (Supplementary Table 1). Analysis was performed using Bio-Rad CFX96.

Statistics & Reproducibility
BRET ratios were defined as the acceptor emission divided by the donor emission. Normalized BRET was determined by subtracting the mean value of the baseline prior to stimulation from all data points. Net BRET was calculated by subtracting the donor only (transfection without HaloTag or YFP) BRET ratio. To determine ligand-induced effects, \u2206BRET was calculated as the difference between vehicle and WNT-treated wells, where each well was normalized to the mean value of the baseline. For analysis of G-CASE experiments, \u2206BRET (%) was calculated for each well as a percent over the baseline ([(BRET ratio (treated) \u2013 mean BRET ratio (baseline))/mean BRET ratio (baseline)] \u00d7 100). Subsequently, the average \u2206BRET (%) of vehicle control was subtracted. Max \u2206BRET (%) was defined as maximum change from the baseline following stimulation in the kinetic experiments. Area under curve (AUC) was determined for 6 minutes post-treatment. Saturation curves were fit using a one site-specific binding model. BRET denotes the maximum net BRET signal. BRET represents the acceptor/donor ratio required to reach the half-maximal BRET signal. Dose-response curves were fit using a nonlinear three-parameter model. For all statistical tests, p<0.05 denoted by one asterisk was considered significant, and normality was confirmed. All data were analyzed with GraphPad Prism, and details of statistical analyses are provided in the source data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Hardy, EP, et al., seeks to demonstrate that the product of the PKD1 gene, polycystin-1/PC1, is a WNT-activated, heterotrimeric G protein-coupled receptor (GPCR) despite its non-canonical 11-transmembrane domain structure. This represents a long-held hypothesis in the PKD research field that was based on previous observations of the binding of multiple Gα families by the PC1 cytosolic C-tail and its G protein-dependent activation of promoter-luciferase reporters, in addition to other studies. More recently, the Tsiokas lab demonstrated binding of WNT ligands to an extracellular domain of PC1 activates the ion channel complex formed by PC1 and PC2 (product of the PKD2 gene). In the current work, the authors use transient transfection of HEK293T cells with various BRET-based G protein binding and activity sensors and NanoLuciferase (Nluc) C-tagged PC1 to show the ability of WNTs 3A, 5A and 9B to result in Gα-Gβγ dissociation of Gαq and Gαi1-3 proteins but not of Gαs or Gα13 in a PC1-dependent manner. WNT9B was further shown to induce binding of Gαq and Gαi1-3 proteins to PC1 and to activate (GDP to GTP exchange) Gαi family members. A variety of pathogenic PKD1 mutations that truncate the C-terminal, cytosolic tail or disrupt PC1 surface expression, WNT binding, GPS cleavage, or G protein signaling were investigated and found to reduce WNT9B-mediated Gαi1-3 -Gβγ dissociation. Using chimeras of Gαi3 and Gαs, the G protein coupling selectivity of PC1 was shown to involve more than the C-terminal helix 5 of Gαi3. To examine a potential role for PC2 in PC1 GPCR function, the authors assess Gαi family dissociation and PC1 cell surface expression (via IF and BRET methods) under conditions of transient, CRISPR-mediated PC2 knockdown (KO) and/or PC2 KO with addback expression of wild type (WT) or mutant (mouse) PKD2 clones that disrupt PC2 channel activity (D509V) or primary cilium trafficking (E442G). These results showed the (known) chaperone function of PC2 was required to enable PC1-mediated activation of Gαi3 and were furthermore interpreted to be independent of PC2 channel activity. Utilizing a cAMP GloSensor assay, the authors attempt to link the PC1 GPCR function with a pathogenic mechanism of ADPKD by showing the ability of WNTs 3A, 5A and 9B to inhibit the accumulation of forskolin-stimulated cytosolic cAMP levels in a manner that was dependent on PC1, Gαi and PC2. Finally, BRET experiments using PKD1-Nluc and GRK- or β-arrestin-HaloTag constructs showed PC1 selectively interacts with GRK2, GRK6 and β-arrestin-2 (ARRB2) in a manner that is dependent on PC2 and promotes PC1 internalization. Based on these data, the authors conclude that PC1 directly and selectively mediates Gαi protein family activation in response to WNT9B which provides a direct link to cAMP metabolism leading them to propose that “impaired PKD1-Gαi1-3 coupling is the root cause of cyst formation in ADPKD.”\n\nThe work presented represents a significant advancement in demonstrating a GPCR function for PC1 using real time assay systems that directly test the ligand-dependent interaction and activation of heterotrimeric G proteins with full-length PC1. Furthermore, the selective activation of Gαi family members by WNT-activated PC1 provides a new pathogenic mechanism for the aberrantly elevated levels of cAMP that are known to drive cystic disease. Finally, the results have potential implications for both the (canonical) GPCR and TRP channel fields.\n\nStrengths include the use of live cell assay systems that directly measure G protein binding and activation, cAMP levels or GRK and β-arrestin binding in a time-dependent manner, the inclusion of important controls to rule out the involvement of endogenous Frizzled or LRP6 receptors, and controls for Gαi-mediated effects by including PTX or GPSM1 (a GDI for Gαi3).\n\nGeneral weaknesses include: all of the experiments are cell culture-based with transient overexpression of PC1/PC2 constructs, and with one exception were all performed in 293T cells; not all of the G proteins were assessed in every assay and in some cases, the inclusion of Gα13 and Gαs as negative controls in some experiments would have strengthened experimental validity. Specific comments and suggestions are listed below.\n\n1) One of the major questions remaining is whether the plasma membrane-localized PC1-PC2 complex is the functional GPCR as opposed to a scenario where PC1 dissociates from PC2 in order to . The studies do not demonstrate that the complex of PC1-PC2 has ‘receptor function’, only that ‘receptor function’ is dependent of PC2 chaperone activity. As such, the manuscript is somewhat inconsistent in regard to how this distinction is handled. There are numerous instances where the writing seems to imply that the PC1-PC2 complex is the functional GPCR. Examples include: line 294- “partially reverted PKD1/PKD2-mediated Gai3 coupling”; line 295- “the kinase activity of GRK6 was essential for its effect on PKD1/PKD2 function”; line 297- “the partial reversal on PKD1/PKD2 function significant”; line332- “directly involving PKD1/PKD2. As the receptor function of the PKD1/PKD2 complex.” Furthermore, the sentence “It would be interesting to know whether the PKD1/PKD2 complex functions as a metabotropic receptor” (lines 337-338) is confusing since, as written, the preceding sentences seem to assume the PKD1/PKD2 complex is the functional form of the GPCR activity. In such instances these referrals to the PKD1/PKD2 complex as the functional GPCR should be revised to assure accurate interpretation of the data and improve clarity.\n\n2) The second major question remaining is whether the ion channel activity of the PC1-PC2 complex is involved in PC1 G protein signaling. Since PKD2-D509V, which lacks channel activity, also prevents trafficking of PC1 to membrane, conclusions regarding a role or necessity for PKD2-mediated “calcium permeation” cannot be made. Furthermore, lines 333-337- “As the receptor function of the PKD1/PKD2 complex does not require Ca2+ permeation, our data are compatible with Cryo-EM data of the PKD1/PKD2 complex, whereby three positively charged amino acid residues of PKD1 occlude the PKD1/PKD2 ionic pore16. It is quite fascinating that while individual PKD1 and PKD2 proteins bear “TRP channel-like” domains, when they form a complex, not only is the channel function blocked, but a GPCR function is enabled” as written is an over-interpretation of the manuscript’s data and therefore should be tempered as a hypothesis and not as a statement of fact.\n\n3) Fig 5- The relevance of PC1-mediated activation of Gαi and the inhibition of cellular cAMP accumulation to PKD pathogenesis would be stronger if the experiments (Fig. 5) had been performed in renal epithelial cell lines with endogenous expression PC1 and null for PC1 or with receptor-mediated stimulation of cAMP rather than forskolin, which may beyond the scope of this manuscript. Similarly, However, the inclusion of the PKD1 mutants that prevent Wnt-binding (W135C) or interfere with G protein signaling (L4132∆) in the experiments examining the effect of WNT9B on cellular cAMP levels could be performed and would strengthen the pathogenic relevance and eliminate the correlative aspects of the finding.\n\n4) The manuscript lacks discussion of the results in consideration of previous literature, e.g., PC1 C-tail-mediated binding/sequestration of Gαs and Gα12 (PMID: 15203210, PMID 20106977, PMID: 29530879) the inhibition of PC1-dependent Gαi signaling by PC2 co-expression (PMID: 11786542), and others (PMID: 14766803).\n\n5) The section on ‘Limitations of the study’ does not appropriately address the shortfalls of the manuscript’s experimental approach and the potential caveats regarding interpretations. In particular, the limitations of the BRET assay, the influence of the cellular context and how this may bias results, and overexpression of components including the likely nonphysiological ratio of PC1 to PC2.\n\n6) Do the levels of WNT9B-induced PKD1-mediated Gαi1-3 dissociation (Max ∆BRET %) differ between the various PKD1 variants in Fig. 1d-g, and if so, what are the potential implications?\n\n7) Line 342- “..proximity to GRK6, whose activity is necessary for internalization and ARRB-2 recruitment“. How was the ARRB2 interaction with PKD1 shown to be dependent on GRK activity? Assessing BRET of ARRB2-HaloTag with S3162A or S3162D mutants of PKD1-Nluc would link the current observations (Fig 6j) with previous studies.\n\n8) As the authors have previously demonstrated that WNTs 3A, 5A, and 9B activate the PC1/PC2 channel activity via binding to PC1, how do they envision that the activation of PC1 GPCR versus ion channel activity is differentiated by these same ligands?\n\n9) For Fig 4c, why doesn’t the re-expression of WT-PKD2 completely restore WNT9B/PKD1-mediated Gai3 dissociation activity, especially as it was noted that PC2 increases overall levels of PKD1? What is the interpretation of the (significant) difference in abilities of PKD2-E442G versus PKD2- D509V to partially rescue Gai3 dissociation? Does the retention of channel activity by E442G suggest some degree of dependence on PKD2 channel activity? Perhaps examining and comparing the extent of rescue by D509V and E442G in IMCD3 cells after allowing ciliation would be a more relevant assessment of the involvement of PKD2 channel activity.\n\n10) Methods- (a) please clearly state the numbers of technical replicates per experiment and the numbers of biological (experimental) repeats for each analysis. (b) What was the range of “the steady-state operating temperature of the microplate reader”? (c) At what residue was PKD2-∆C truncated (i.e., does it completely lack the C-tail)? What is literature reference for this clone or its method of construction?\n\n11) Western blot images should indicate locations of molecular weight marker bands (not just ~MW of band detected).\n\n12) Lines 128-129: “WNTs did not induce Gα dissociation for any of the six subtypes in mock-transfected cells (Supplementary Fig. 1e).” statement should be qualified for Gai1- and i2-CASE as the ∆BRET (%) levels reached for some or all of the WNTs tested were similar to the levels observed with PC1 (Fig 1a). How were these ∆BRET (%) levels determined to be statistically different?\n\n13) Lines 101-102- “and pcDNA3 (to assess endogenous GPCR activation in response to WNT) or PKD1 (Fig. 1a)” is missing referral to ‘Supplementary Fig. 1e’ for the pcDNA3 experiments.\n\n14) Supp Fig 1c schematic shows PC1 with a C-terminal Nluc tag rather than an eGFP tag.\n\n15) Supp Fig 2a & b- The Western blot should be labeled for the various forms of PC1 detected (i.e., FL, NTF, CTF). In Supp Fig 2b, the band migrating between 150 and 250 kDa (whose intensity increases the most upon mouse PKD2 co-expression) has a larger than expected apparent molecular weight for the CTF form of PC1. A similar aberrant migration observation can be made for the presumptive CTF bands in the α-HA blot in part (a). What is the explanation for this?\n\n16) Line 226, “possibly via protein-protein-mediated stabilization” should cite prior studies in support of this interpretation.\n\n17) Lines 226-227, ‘4e’ should be removed from “PKD2-E442G showed reduced stabilization of PKD1 compared to wild-type PKD2 (Fig. 4e and g)”.\n\n18) Supp Fig 5- The sentence: “residual PKD2 signal (lane 4) is obtained from untransfected cells (transfection efficiency, 80-90%)” is confusing and should be rewritten to increase clarity.\n\n19) The legend for Fig 6h mentions Gαi1-CASE but the Results section indicates Gαi3. Fig 6k shows ARRB2 bound to second intracellular loop of PC1 rather than its mapped location within first loop.", "reviewer_2": "The manuscript submitted by Hardy et al. entitled: “A heteromeric TRP channel functions as a WNT-activated G protein-coupled receptor” is quite an intriguing study with some content that can be best described as ”out of the box thinking” but is a very well controlled study.\n\nThe abstract and introduction are well written and the proposed hypothesis is thoroughly tested with different approaches. I enjoyed reading the manuscript since it logically build.\n\nNonetheless I have a few points that were unclear while reading the manuscript:\n\nIn the results section the authors sate:\nLine 175\n“In contrast to PKD1, Gαi-coupled CaSR and Gαs-coupled β2AR each showed a complete exchange in coupling selectivity correlating with the swap in Gα C-terminal domain (Fig. 2f and 2g), thus validating the functionality of the chimeras.\nCompared to the CaSR and β2AR, our results for PKD1 indicate that the Gα C-terminus contributes only partially to selective coupling and suggests additional/multiple interactions between PKD1 and the G protein may dictate selectivity. Thus, the mechanisms dictating G protein coupling selectivity of PKD1 is different from traditional 7-TM containing GPCRs.”\n\nThis coupling selectivity might indeed be different but the reason is not clear and fully proven.\n\nTherefore I would suggest a minor addition to rather state:\nThus, the mechanisms dictating G protein coupling selectivity of PKD1 is likely different from traditional 7-TM containing GPCRs\n\nNext point:\nLine 222\n“Interestingly, PKD2-E442G also failed to target PKD1-eGFP to the plasma membrane (Fig. 4e, lower panels). PKD1-Nluc co-localization with HaloTag-CAAX quantitatively supported the live cell imaging data (Fig. 4g).”\n\nHow was this experimental part quantified to allow the statement? I did not see this… Higher BRET values with a CAXX tagged protein could also occur by better trafficking or folding. This does not necessarily need to be due to higher expression. Maybe I did miss something?\n\nDiscussion\nLine 339\n…”PKD1 does not only mediate ligand-dependent Gα-mediated signaling, but also GRK/ARRB mediated recruitment and internalization. This is another defining property of all known GPCRs that PKD1 fulfills”.\n\nI think this statement is wrong in two points:\nI did not see any data that allow conclusion internalization. GRK and ARRB yes but internalization was not tested by the authors. It`s likely but no data are shown to support such statement.\nSecond, certainly not all GPCRs are regulated by GRK and ARRB (see PMID: 33472843).\n\nLine 351\n“We show that PKD1 is a unique type of GPCR containing 11 rather than 7 TM segments typically 351 seen in classical GPCRs. Notably, PKD1 has a distinct sequence and structure, not simply an 352 additional extension of the “typical 7-TM protein” domain seen in canonical GPCRs. This 353 unexpected result expands the general signaling paradigm for the GPCR group of cell surface 354\nreceptors. The unique structural features and functional properties of the Polycystin complex 355 described here lead us to propose that the Polycystins (PKD1 and PKD2) and possibly Polycystin-356 like molecules (i.e., PKD1-L (1-3), PKD2-L (1-3)) form a new class of GPCRs.”\n\nIn the absence of any structural information people in the GPCR field might find this very provocative. I think the authors should be encouraged to look into the May issue 2025 of Nature Chem Biol Vol 32 No5 and cite the two papers on LYCHOS by Xiong et al. or Zhao et al.\n\nBoth studies showing structural data of the multi TM domain protein LYCHOS with an integrated GPCR/7TM fold that looks like a GPCR as we are used to… This might take out a little bit of the discussion…\n\nOverall I must say this paper is very well controlled and experimentally designed. I am sorry that I took so long to review it…"}, "author_response": "We thank the reviewers for constructive criticisms and suggestions for improvement. Please see\nbelow our responses to points raised.\n\nReviewer #1 (Remarks to the Author):\n[...]\nResponse\nPlease see new Fig. 8e-h. It is difficult to envision that PKD2 chaperones PKD1 to the cell\nsurface and then it dissociates from it. Structural studies show a stable PKD1/PKD2\ncomplex with 1:3 stoichiometry1. We provide new evidence that PKD1 or PKD2 are\ninternalized and accumulate in the early endosome via similar kinetics in response to WNT-\n9B. As only activated PKD1/PKD2 should follow that route, our new data provide compelling\nevidence that PKD1 and PKD2 remain associated at the plasma membrane during\nactivation by the WNT-9B ligand. We revised the text to clarify that the PKD1/PKD2 complex\nrather than PKD1 alone functions as a GPCR. To avoid confusion, we removed the sentence\nabout testing whether PKD1/PKD2 is a metabotropic receptor. We meant to say that it would\nbe interesting to know whether the channel activity of the PKD1/PKD2 complex is\ndownstream of G protein activation (please see lines 434-437).\n\n2) The second major question remaining is whether the ion channel activity of the PC1/PC2\ncomplex is involved in PC1 G protein signaling. Since PKD2-D509V, which lacks channel\nactivity, also prevents trafficking of PC1 to membrane, conclusions regarding a role or\nnecessity for PKD2-mediated “calcium permeation” cannot be made. Furthermore, lines\n333-337- “As the receptor function of the PKD1/PKD2 complex does not require Ca2+\npermeation, our data are compatible with Cryo-EM data of the PKD1/PKD2 complex,\nwhereby three positively charged amino acid residues of PKD1 occlude the PKD1/PKD2\nionic pore16. It is quite fascinating that while individual PKD1 and PKD2 proteins bear “TRP\nchannel-like” domains, when they form a complex, not only is the channel function blocked,\nbut a GPCR function is enabled” as written is an over-interpretation of the manuscript’s data\nand therefore should be tempered as a hypothesis and not as a statement of fact.\n\nResponse\nPlease see new data in Fig. 5a-c. We provide new data using the PKD2-W201A and PKD2-\nF604P to show that PKD2 variants with reduced2 or enhanced3 channel activity, respectively,\ndid not affect the PKD1/PKD2 function as a GPCR. We also treated cells with La3+ prior to\nligand addition, which blocks the cation permeability of PKD1/PKD2 or PKD2, and found that\nactivation of the GPCR function of PKD1/PKD2 by WNT-9B was not affected. These data\nlead us to conclude that the channel activity of PKD1/PKD2 is independent of its GPCR\nactivity.\n\n3) Fig 5- The relevance of PC1-mediated activation of Gαi and the inhibition of cellular cAMP\naccumulation to PKD pathogenesis would be stronger if the experiments (Fig. 5) had been\nperformed in renal epithelial cell lines with endogenous expression PC1 and null for PC1 or\nwith receptor-mediated stimulation of cAMP rather than forskolin, which may beyond the\nscope of this manuscript. Similarly, However, the inclusion of the PKD1 mutants that prevent\nWnt-binding (W135C) or interfere with G protein signaling (L4132∆) in the experiments\nexamining the effect of WNT9B on cellular cAMP levels could be performed and would\nstrengthen the pathogenic relevance and eliminate the correlative aspects of the finding.\n\nResponse\nPlease see new data in Fig. 6f and h. We followed the suggestion of the reviewer and tested\nthe pathogenic mutations recommended for their effects on WNT-9B-induced reduction of\ncAMP.\n\nIn regard to testing the functionality of endogenous PKD1 as a GPCR, we provide new data\nsupporting this statement in new Fig. 1j and k. We believe that this is a major improvement\nof the manuscript as it shows that endogenous PKD1 is coupled to G activation.\ni3\n\n4) The manuscript lacks discussion of the results in consideration of previous literature, e.g.,\nPC1 C-tail-mediated binding/sequestration of Gαs and Gα12 (PMID: 15203210, PMID\n20106977, PMID: 29530879) the inhibition of PC1-dependent Gαi signaling by PC2 co-\nexpression (PMID: 11786542), and others (PMID: 14766803).\n\nResponse\nDiscussion was added based on the recommended references (please see lines 402-421).\n\n5) The section on ‘Limitations of the study’ does not appropriately address the shortfalls of the\nmanuscript’s experimental approach and the potential caveats regarding interpretations. In\nparticular, the limitations of the BRET assay, the influence of the cellular context and how\nthis may bias results, and overexpression of components including the likely\nnonphysiological ratio of PC1 to PC2.\n\nResponse\nDiscussion was modified to include limitations brought up by the reviewer (please see lines\n462-469).\n\n6) Do the levels of WNT9B-induced PKD1-mediated Gαi1-3 dissociation (Max ∆BRET %) differ\nbetween the various PKD1 variants in Fig. 1d-g, and if so, what are the potential\nimplications?\n\nResponse\nFig. 1d-g does not show effects of WNT-9B on G dissociation using different PKD1\ni1-3\nvariants. These effects are shown in new Fig. 2 and their implications on disease\npathophysiology were discussed.\n\n7) Line 342- “..proximity to GRK6, whose activity is necessary for internalization and ARRB-2\nrecruitment“. How was the ARRB2 interaction with PKD1 shown to be dependent on GRK\nactivity? Assessing BRET of ARRB2-HaloTag with S3162A or S3162D mutants of PKD1-\nNluc would link the current observations (Fig 6j) with previous studies.\n\nResponse\nPlease see new Fig. 8d. Pharmacologic inhibition of GRK6 reduced ARRB-2 recruitment.\nWe acknowledge the fact that the effects were marginal, but these inhibitors are toxic and\nthus, they were applied for a short time (up to an hour) at doses that could be barely\ntolerated.\n\n8) As the authors have previously demonstrated that WNTs 3A, 5A, and 9B activate the\nPC1/PC2 channel activity via binding to PC1, how do they envision that the activation of\nPC1 GPCR versus ion channel activity is differentiated by these same ligands?\n\nResponse\nIn our previous work, we showed that WNT-3A, 5A, or 9B bound to PKD1 and activated the\nchannel activity of the PKD1/PKD2 complex without significant differences, i. e., all ligands\ninduced stable whole cell La3+-inhibitable currents with a characteristic 1 min delay after\nligand addition4. Consistently, here, we show that all three ligands activated the same subset\nof G subunits to a similar extend relative to each other (ie, G >G >G ≥G ). G and\ni3 i2 i1 q s\nG were not activated by none of the three ligands. We propose that GPCR activity\n13\nprecedes channel activation, as it happens faster than channel activation. It is conceivable\nthat dissociated G and/or G subunits following ligand activation activate the PKD1/PKD2\nchannel. We are working on addressing this hypothesis.\n\n9) For Fig 4c, why doesn’t the re-expression of WT-PKD2 completely restore WNT9B/PKD1-\nmediated Gai3 dissociation activity, especially as it was noted that PC2 increases overall\nlevels of PKD1?\n\nResponse\nAs we discussed in the text (please see lines 402-421), a critical balance of PKD1, PKD2,\nG subunits, and possibly other signaling components may be needed for full rescues. Re-\nexpression of PKD2 may exceed the optimal levels needed for the formation of functional\ncomplexes with PKD1. In such a case, formation of suboptimal complexes could occur that\ncould facilitate the trafficking of PKD1 to the cell surface, but not its function as a GPCR.\nStructural studies show that the most stable complexes of PKD1/PKD2 are of 1:3\nstoichiometry1. However, forced expression of PKD1 or PKD2 can lead to unstable, sub-\noptimal complexes with a 2:2 stoichiometry, which have been visualized using Atomic Force\nMicroscopy (AFM)5, that could efficiently traffic to the cell membrane but may not support\nfunction. While this is a pure speculation, previous studies have shown that overexpression\nof PKD2 or PKD1 in transgenic mice lead to cystogenesis6 indicating that a critical balance\nof PKD1 and PKD2 is essential for preventing cystogenesis. These possibilities are now\ndiscussed in the manuscript. Technical nuances may also be at play. For example, as we\nshow in Supplementary Fig. 3a, the ratio of PKD1 to the G-CASE sensor is extremely\nimportant for functional coupling. When PKD2 is overexpressed, there is massive\nupregulation of PKD1, which may require re-calibration of the amount of G-CASE sensor\nused.\n\nWhat is the interpretation of the (significant) difference in abilities of PKD2-E442G versus\nPKD2- D509V to partially rescue Gai3 dissociation? Does the retention of channel activity by\nE442G suggest some degree of dependence on PKD2 channel activity? Perhaps examining\nand comparing the extent of rescue by D509V and E442G in IMCD3 cells after allowing\nciliation would be a more relevant assessment of the involvement of PKD2 channel activity.\n\nResponse\nIt is very difficult to know what is more impactful in terms of function, the failure to assemble\na functional complex (E442G) or the failure to target PKD1 to the plasma membrane\n(D509V). Our experiments using variants of PKD2 with reduced or enhanced channel\nfunction without compromising assembly or targeting to the cell surface lead us to propose\nthat the effect of channel activity per se in the GPCR function of PKD1/PKD2 is minimal, if\nany.\n\n10) Methods- (a) please clearly state the numbers of technical replicates per experiment and the\nnumbers of biological (experimental) repeats for each analysis. (b) What was the range of\n“the steady-state operating temperature of the microplate reader”? (c) At what residue was\nPKD2-∆C truncated (i.e., does it completely lack the C-tail)? What is literature reference for\nthis clone or its method of construction?\n\nResponse\n(a) Only biological or experimental replicates were used throughout the manuscript and the\nnumbers are indicated in the legends.\n(b) Experiments using G-CASE sensors, ONE-GO sensors, and G protein recruitment to\nPKD1 were done at 27ºC; experiments used to determine cell surface experiments (ie,\nHaloTag-CAAX) and complex assembly were done at room temperature.\n(c) The PKD2C construct does not include any of the C-terminal tail of PKD2. It truncates\nimmediately after the sixth transmembrane segment of PKD2.\nThis information is now added in the “Methods” section (please see lines 503-504 and 578-\n580).\n\n11) Western blot images should indicate locations of molecular weight marker bands (not just\n~MW of band detected).\n\nResponse\nDone.\n\n12) Lines 128-129- “WNTs did not induce Gα dissociation for any of the six subtypes in mock-\ntransfected cells (Supplementary Fig. 1e).” statement should be qualified for Gai1- and i2-\nCASE as the ∆BRET (%) levels reached for some or all of the WNTs tested were similar to\nthe levels observed with PC1 (Fig 1a). How were these ∆BRET (%) levels determined to be\nstatistically different?\n\nResponse\nIn the revised version, we compared responses among different treatments using the area\nunder the curve (AUC) up to 6 min after the addition of a ligand. This is a more unbiased\napproach, especially for small responses. However, it is very difficult to reliably quantify\nsmall responses shown in Supplementary Fig. 1e.\n\n13) Lines 101-102- “and pcDNA3 (to assess endogenous GPCR activation in response to WNT)\nor PKD1 (Fig. 1a)” is missing referral to ‘Supplementary Fig. 1e’ for the pcDNA3\nexperiments.\n\nResponse\nThis is corrected.\n\n14) Supp Fig 1c schematic shows PC1 with a C-terminal Nluc tag rather than an eGFP tag.\n\nResponse\nThis is correct. The schematic (Supp Fig 1c) shows the tagged constructs used for the\nBRET saturation assay shown in Supp Fig 1d.\n\n15) Supp Fig 2a & b- The Western blot should be labeled for the various forms of PC1 detected\n(i.e., FL, NTF, CTF). In Supp Fig 2b, the band migrating between 150 and 250 kDa (whose\nintensity increases the most upon mouse PKD2 co-expression) has a larger than expected\napparent molecular weight for the CTF form of PC1. A similar aberrant migration observation\ncan be made for the presumptive CTF bands in the α-HA blot in part (a). What is the\nexplanation for this?\n\nResponse\nWe marked the migration of full length PKD1, NTF, and CTF in Supplementary Fig. 2a and\nb. However, it is very difficult to distinguish partially glycosylated full length PKD1 from\nglycosylated NTF, as they run at the same size.\nSupplementary Fig. 2a was done using a 6% SDS-PAGE to separate large molecular weight\nfragments, i.e., full length and NTF. In this case, the CTF runs very close to 150 KDa\n(Supplementary Fig. 2a, lower panel). In Supplementary Fig. 2b, an 8% SDS-PAGE gel was\nused to resolve smaller C-terminal fragments, hence the apparent discrepancy in the size of\nthe CTF (~150-170 KDa). It should be noted that the PKD1-3xHA wild type construct used in\nall experiments (Supplementary Fig. 2a lanes 2 and 9; and Supplementary Fig. 2b), was\nused in a previous study to rescue cystogenesis in Pkd1-null mice7.\n\n16) Line 226- “possibly via protein-protein-mediated stabilization” should cite prior studies in\nsupport of this interpretation.\n\nResponse\nWe are not aware of previous studies reporting the stabilization of full length PKD1 by\nPKD2, except for a 1997 study using C-terminal tails of PKD1 and PKD28. This is now cited.\n\n17) Lines 226-227- ‘4e’ should be removed from “PKD2-E442G showed reduced stabilization of\nPKD1 compared to wild-type PKD2 (Fig. 4e and g)”.\n\nResponse\nDone\n\n18) Supp Fig 5- The sentence: “residual PKD2 signal (lane 4) is obtained from untransfected\ncells (transfection efficiency, 80-90%)” is confusing and should be rewritten to increase\nclarity.\n\nResponse\nPlease see revised legend for Supplementary Fig. 5.\n\n19) The legend for Fig 6h mentions Gαi1-CASE but the Results section indicates Gαi3. Fig 6k\nshows ARRB2 bound to second intracellular loop of PC1 rather than its mapped location\nwithin first loop.\n\nResponse\nPlease see revised legend for new Fig. 7h.\nPlease see revised cartoon (new Fig. 8h).\n\nReviewer #2 (Remarks to the Author):\n[...]\nResponse\nDone. Please see line 203.\n\nNext point:\nLine 222- “Interestingly, PKD2-E442G also failed to target PKD1-eGFP to the plasma\nmembrane (Fig. 4e, lower panels). PKD1-Nluc co-localization with HaloTag-CAAX quantitatively\nsupported the live cell imaging data (Fig. 4g).” How was this experimental part quantified to\nallow the statement? I did not see this… Higher BRET values with a CAXX tagged protein could\nalso occur by better trafficking or folding. This does not necessarily need to be due to higher\nexpression. Maybe I did miss something?\n\nResponse\nIn new Fig. 5e (old Fig. 4e), we are comparing steady-state BRET between PKD1-Nluc and\nHaloTag-CAAX in the presence of wild type PKD2 or PKD2-E442G. As you can see, BRET\nlevels are reduced when PKD2-E442G is expressed compared to wild type PKD2. We interpret\nthese data as reduced expression of PKD1-Nluc to the cell surface.\n\nDiscussion\nLine 339- …”PKD1 does not only mediate ligand-dependent Gα-mediated signaling, but also\nGRK/ARRB mediated recruitment and internalization. This is another defining property of all\nknown GPCRs that PKD1 fulfills”. I think this statement is wrong in two points: I did not see any\ndata that allow conclusion internalization. GRK and ARRB yes but internalization was not tested\nby the authors. It’s likely but no data are shown to support such statement. Second, certainly not\nall GPCRs are regulated by GRK and ARRB (see PMID: 33472843).\n\nResponse\nPlease see new Fig. 8d-h. We added new data showing that PKD1/PKD2 is internalized in\nresponse to WNT-9B, as determined by the time-dependent departure from the plasma\nmembrane and accumulation in the early endosome. We also provide new data showing that\nremoval from the plasma membrane is inhibited by barbadin, an ARRB inhibitor (Fig. 8g), and\nWNT-9B-induced recruitment of ARBB2 to PKD1 is partially suppressed by a brief incubation of\nGRK6 inhibitors (Fig. 8d). As per the reviewer’s recommendation, we removed the sentence in\nthe discussion to avoid generalizations about the regulation of all GPCRs by GRK/ARRB.\n\nLine 351- “We show that PKD1 is a unique type of GPCR containing 11 rather than 7 TM\nsegments typically 351 seen in classical GPCRs. Notably, PKD1 has a distinct sequence and\nstructure, not simply an 352 additional extension of the “typical 7-TM protein” domain seen in\ncanonical GPCRs. This 353 unexpected result expands the general signaling paradigm for the\nGPCR group of cell surface 354 receptors. The unique structural features and functional\nproperties of the Polycystin complex 355 described here lead us to propose that the Polycystins\n(PKD1 and PKD2) and possibly Polycystin-356 like molecules (i.e., PKD1-L (1-3), PKD2-L (1-3))\nform a new class of GPCRs.”\n\nIn the absence of any structural information people in the GPCR field might find this very\nprovocative. I think the authors should be encouraged to look into the May issue 2025 of Nature\nChem Biol Vol 32 No5 and cite the two papers on LYCHOS by Xiong et al. or Zhao et al.\n\nBoth studies showing structural data of the multi TM domain protein LYCHOS with an integrated\nGPCR/7TM fold that looks like a GPCR as we are used to… This might take out a little bit of the\ndiscussion…\n\nResponse\nWe included in the Discussion the recommended articles on LYCHOS. Please see lines 454-\n456.\n\nOverall I must say this paper is very well controlled and experimentally designed. I am sorry that\nI took so long to review it…"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript by Hardy, EP, et al., has adequately addressed this reviewer's major concerns. New data are included that provide additional support for the PKD1/PKD2 complex as the functional form for its WNT-mediated GPCR activity, demonstrate PKD1/PKD2 GPCR activity is independent of PKD2 channel function, and strengthen the relevance of PKD1-mediated Gαi activation with PKD pathogenesis. Additional points of concern were also addressed through modification to the text. This manuscripts represents a significant contribution to our understanding of PKD1 function and to the GPCR field.", "reviewer_2": "The authors have addressed my questions and I have no more to say than congratulation on this nice piece of research."}, "author_response": ""}]

Decision letter