phosphorylation in bovine adrenal cortex-derived endothelial cells [14]. It has also been shown that PK1 suppressed giant contraction in the circular muscles of mouse colon, and that this effect was blocked by the eNOS inhibitor L-NAME. In vitro, PK1 stimulated the release of NO from longitudinal musclemyenteric plexus cultures [84]. We have found that PK1 treatment elevated eNOS mRNA levels in luteal endothelial cells. Cells were also treated in the presence of PI3/Akt pathway inhibitor, which caused a 20– 40% reduction in eNOS levels (Levit and Meidan, unpublished data). These opposing effects of Indinavir and PK1 on eNOS levels and NO production/release are compatible with the chemically based hypothesis arising from the current work, which suggests that Indinavir can bind to the hPKR subtypes by acting as a PKR antagonist. We suggest that this would subsequently reduce eNOS expression levels in endothelial cells and impair NO bioavailabil- ity, leading, at least partially, to the observed Indinavir side effects in HIV patients. This hypothesis should be explored experimen- tally in future studies to determine the possible binding of Indinavir to hPKRs and its subsequent effects. The proposed hypothesis is in accordance with the concept of polypharmacology - specific binding and activity of a drug at two or more molecular targets, often across target boundaries. For example, ligands targeting aminergic family A GPCRs were also found to act on protein kinases [85]. These "off-target" drug actions can induce adverse side effects and increased toxicity. In contrast, there are also cases where the drug is a "magic shotgun", and its clinical effect results from its action on many targets, which in turn enhances its efficacy. For example, drugs acting through multiple GPCRs have been found to be more effective in treating psychiatric diseases such as schizophrenia and depression [86]. This concept was demonstrated by Keiser and colleagues [48] who utilized a statistics-based chemoinformatics approach to predict off-targets for ,900 FDA-approved small-molecule drugs and ,2800 pharmaceutical compounds. The targets were compared by the similarity of the ligands that bind to them. This comparison resulted in 3832 predictions, of which 184 were inspected by literature searches. Finally, the authors tested 30 of the predictions experimentally, by radioligand competition binding assays. For example, the a1 adrenergic receptor antagonist Doralese was predicted and observed to bind to the dopamine D4 receptor (both are aminergic GPCRs), and most interestingly, the HIV-1 reverse transcriptase inhibitor Rescriptor was found to bind to the histamine H4 receptor. The latter observation crosses major target boundaries. These two targets have neither an evolutionary or functional role nor structural similarity in common. However, some of the known side effects of Rescriptor treatment include painful rashes. This observation is similar to our findings of possible interactions of Indinavir and the other enzyme-targeting VLS hits with the PKR subtypes. In summary, defining the selective and non-selective actions of GPCR targeting drugs will help in advancing our understanding of the drugs’ biological action and the observed clinical effect, including side effects. Potential differences between the hPKR subtypes Both subtypes are capable of binding the cognate ligands at approximately the same affinity [12]. Therefore, the diversification of cellular events following activation of the subtypes [16] is not likely to stem from the extracellular loop regions. This suggestion warrants further experimental investigation. Our study also suggests, in agreement with previous findings, that small-molecule antagonists are not likely to easily differentiate between the subtypes. This is because the TM-bundle small-molecule binding Small-Molecule Binders of Prokineticin Receptors site identified in this study is identical in its amino acid composition for the two hPKR subtypes. Thus, an intriguing question arises: what molecular mechanisms are responsible for PKRs’ differential signaling patterns? The variation of protein amino acid composition in the extracellular and intracellular regions of PKRs is significant (represented as black-filled circles in Fig. 1). Moreover, analysis of the level of selection acting on the two PKR subtypes, by calculating the ratio between non-synonymous (Ka) and synony- mous (Ks) substitutions [46,47] predicted purifying selection for the transmembrane helices of both subtypes (figure S7). This analysis should be expanded in future studies, as PKR subtype sequences from additional species become available. The variation in amino acid composition in the intracellular regions of the PKR subtypes may affect at least two signaling events: receptor phosphorylation by kinases and the receptors’ coupling to G proteins. We therefore suggest that this region is most likely to be involved in differential signaling, as detailed next. Interaction with G proteins Differential coupling of PKR subtypes to G proteins has been demonstrated experimentally (reviewed in [16]). Coupling of PKR1 to Ga11 in endothelial cells induces MAPK and PI3/Akt phosphorylation, which promotes endothelial cell proliferation, migration and angiogenesis [11]. In cardiomyocytes, coupling of PKR1 to Gaq/11 induces PI3/Akt phosphorylation and protects cardiomyocytes against hypoxic insult. In contrast, PKR2 couples to Ga12 in endothelial cells, causing Ga12 internalization and down-regulation of ZO-1 expression, leading to vacuolarization and fenestration of these cells. In cardiomyocytes, PKR2 acts through Ga12 and Gaq/11 coupling and increases cell size and sarcomere numbers, leading to eccentric hypertrophy [16]. Thus, sites of interactions with G-proteins may represent an additional factor affecting PKR subtype specificity. Receptor Phosphorylation It is well established that GPCR phosphorylation is a complex process involving a range of different protein kinases that can phosphorylate the same receptor at different sites. This may result in differential signaling outcomes, which can be tailored in a tissue- specific manner to regulate biological processes [87]. We suggest that part of the differential signaling of PKR subtypes may be due to differential phosphorylation of the intracellular parts of the receptors. Namely, phospho-acceptor sites may be missing in one subtype or another, and analogous positions may be phosphory- lated by different kinases due to variation in the positions surrounding the phospho-acceptor residue (which is conserved between subtypes), thus, changing the kinase recognition sequence [88]. Hence, using different combinations of kinases for each subtype results in different phosphorylation signatures. This phosphorylation signature translates to a code that directs the signaling outcome of the receptor. This may include two types of signaling events: (a) common phosphorylation events for both subtypes will mediate common regulatory features such as arrestin recruitment and internalization and (b) subtype-specific events will mediate specific signaling functions related to the specialized physiological role of the receptor subtype. Preliminary analysis using prediction tools for phosphorylation sites suggests that Thr178 (Thr169) in the second intracellular loop and Tyr365 (Gln356) in the cytoplasmic tail of hPKR1 (hPKR2) may represent subtype-specific phosphorylation-related sites (Levit, Meidan and Niv, unpublished data). Further experimental studies are required to elucidate the role of receptor phosphorylation in specific signaling events following activation of PKR subtypes. PLoS ONE | www.plosone.org 14 November 2011 | Volume 6 | Issue 11 | e27990