Sensing and transduction of acid‐base disturbances by receptor protein tyrosine phosphatase γ
Authors: Moss Fraser J. , Wass Amanda B. , Watterson Summer J. , Boron Walter F. 
Published: June 2021

Basolateral [CO2] and [HCO3−], but not extracellular pH (pHo), acutely regulates HCO3− reabsorption (JHCO3) from renal proximal tubules (PT). Receptor protein tyrosine phosphatase γ (RPTPγ) is normally present in the PT basal membrane. In RPTPγ knockout mice, JHCO3 is not sensitive to changes in basolateral [CO2] and [HCO3−]. Furthermore, RPTPγ knockouts lack the normal defense to whole‐body metabolic acidosis (MAc). RPTPγ monomers possesses two phosphatase domains (D1 & D2) in their intracellular region, though the D2 domain is catalytically inactive. Upon dimerization, the D2 domain of one protomer occludes the active site of the D1 domain of the second protomer. The extracellular region of RPTPγ possesses a carbonic anhydrase (CA) like domain (CALD), whose structure is strikingly similar to α‐CAs but which lacks the amino acid residues necessary for CA activity. We hypothesize that although the CALD is incapable of interconverting CO2 and HCO3− it can still bind CO2 or HCO3−. In addition, we hypothesize that the identity of the ligand bound to the extracellular CALD favors either dimerization or monomerization of the intracellular RPTPγ phosphatase domains to modulate the protein's phosphatase activity. To detect the interaction of two RPTPγ monomers, we fuse RPTPγ with the pH‐ and halide‐insensitive GFP variants Aquamarine (Aq) to serve as a Förster resonance energy transfer (FRET) donor, and Citrine (Cit) to serve as a FRET acceptor, and co‐express the fusion proteins in HEK293 cells. We subject the cells to a variety of acid‐base disturbances for 5 min at 37°C, and then fix in 4% PFA (20 min at 4°C). We acquire data from co‐expressing cells, normalizing FRET for donor and acceptor expression levels (NFRET). MAc (5% CO2/11 mM HCO3−/pH 7.1) produces a small, non‐significant reduction in NFRET, compared to control (5% CO2/22 mM HCO3−/pH 7.40), but metabolic alkalosis (MAlk; 5% CO2/44 mM HCO3−/pH 7.7) doubles NFRET. When we increase [CO2]o from 2.5% (respiratory alkalosis; RAlk) to 5% (Ctrl) to 10% (respiratory acidosis; RAc), NFRET decreases. In parallel experiments, we co‐transfect cells with RPTPγ‐Aq and Citrine‐fused ErbB1 (a candidate downstream target for RPTPγ; ErbB1‐Cit). Both MAc and RAc substantially increase RPTPγ/ErbB1 NFRET whereas MAlk and RAlk result in small NFRET reductions. Together, our data suggest that HCO3− and CO2 compete for CALD binding to control the RPTPγ dimerization state (and, presumably, phosphatase activity) and while RPTPγ maintains a basal interaction with ErbB1, raising [CO2]o enhances RPTPγ/ErbB1 interactions consistent with our observation that raising [CO2]o tends to decrease RPTPγ/RPTPγ interactions. We are currently investigating both the time‐course of RPTPγ dimerization events and the interactions of RPTPγ with downstream signaling molecules upon initiation of an acid‐base disturbance in the local environment.Support or Funding InformationNIH R01 DK113197This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.