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A Novel GPCR in the Renal Proximal Tubules, Gprc5c, Is Involved in Systemic pH Regulation
Author(s) -
Rajkumar Premraj,
Cha Boyoung,
Donowitz Mark,
Hirabayshi Yoshio,
Pluznick Jennifer L
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.967.3
Subject(s) - g protein coupled receptor , internalization , kidney , receptor , hek 293 cells , microbiology and biotechnology , chemistry , ligand (biochemistry) , transfection , biology , biochemistry , endocrinology , gene
Recent studies have found that novel G‐protein coupled receptors (GPCRs) are utilized as specialized chemosensors to detect metabolites in a variety of tissues, and are often involved in mediating essential physiological processes and metabolic pathways. Our lab aims to uncover the functional role of novel GPCRs in the kidney, and to that end we recently used a Taqman‐based mouse GPCR array to identify GPCRs with unknown function that are expressed at high levels in murine kidney (Rajkumar, et al PLoS One 2014). In this study, we focused on elucidating the functional role of one such novel GPCR, Gprc5c. Gprc5c is an orphan GPCR with no known ligand which we found to be present in the kidney by real‐time PCR at a comparable level to the well‐studied and highly expressed Angiotensin receptor, AT1a (ΔCt AT1a : 6.25±0.24; ΔCt Gprc5c : 7.0±0.16). Using immunohistochemistry, we found that Gprc5c localizes to the renal proximal tubule apical brush border in wild‐type (WT) mice, where it colocalized with both LTA (lotus tetragonolobus agglutinin) and megalin (Gprc5c staining was absent in whole‐animal Gprc5c knockout (KO) mice). Because Gprc5c is an orphan receptor with no known ligand, we performed ligand‐induced GPCR internalization assays in vitro to identify activators of Gprc5c in transiently transfected HEK293T cells. We found that alkaline pH triggered Gprc5c activation, with maximal internalization seen above pH 7.4 (pH 8.0>pH 7.4>pH 6.5). Importantly, other GPCRs tested as controls showed no changes in internalization over the pH range tested. In agreement with a role for Gprc5c in responding to changes in extracellular pH, we found that Gpr5c KO mice have alkaline urine pH compared to wild‐type littermates (WT: 5.5±0.02, n=4; KO: 5.76±0.05, n=5; p<0.05). In addition, preliminary studies indicate that the plasma pH of Gprc5c knockout mice trends mildly acidic (WT: 7.44±0.04, n=4; KO: 7.34±0.02, n=4; p=0.07). To begin to understand the mechanism by which Gprc5c may modulate pH regulation, we loaded HEK293T cells with BCECF‐AM and determined that Gprc5c increases the activity and set point of NHE3 (a sodium‐hydrogen exchanger expressed in the brush border of the renal proximal tubule). Under alkaline conditions (pH8), we find that Gprc5c increases sodium proton exchange by 1.6 fold compared to control cells (p<0.05), and that this increase in activity is Tenapanor‐sensitive, implying that it is mediated via NHE3. Together, these results demonstrate that Gprc5c localizes to the renal proximal tubule where it contributes to pH homeostasis via modulation of NHE3 function.