Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

Abstract

Edited by Henrik G. DohlmanGPRC6A is a G protein-coupled receptor activated by Lamino acids, which, based on analyses of knock-out mice, has been suggested to have physiological functions in metabolism and testicular function. The human ortholog is, however, mostly retained intracellularly in contrast to the cell surface-expressed murine and goldfish orthologs. The latter orthologs are G q -coupled and lead to intracellular accumulation of inositol phosphates and calcium release. In the present study we cloned the bonobo chimpanzee GPRC6A receptor, which is 99% identical to the human receptor, and show that it is cell surface-expressed and functional. By analyses of chimeric human/mouse and human/bonobo receptors, bonobo receptor mutants, and the single nucleotide polymorphism database at NCBI, we identify an insertion/deletion variation in the third intracellular loop responsible for the intracellular retention and lack of function of the human ortholog. Genetic analyses of the 1000 genome database and the Inter99 cohort of 6,000 Danes establish the distribution of genotypes among ethnic groups, showing that the cell surface-expressed and functional variant is much more prevalent in the African population than in European and Asian populations and that this variant is partly linked with a stop codon early in the receptor sequence (rs6907580, amino acid position 57). In conclusion, our data solve a more than decade-old question of why the cloned human GPRC6A receptor is not cell surface-expressed and functional and provide a genetic framework to study human phenotypic traits in large genome sequencing projects linked with physiological measurement and biomarkers.Communication between the exterior and interior of cells is essential for cellular survival. A pivotal class of proteins, specialized to carry out such signal transduction, is the G proteincoupled receptors (GPCRs), 2 a family that includes ϳ800 subtypes in humans. The GPCR class C, group 6, member A (GPRC6A) belongs to the non-olfactory GPCRs as part of the class C receptors. Human GPRC6A (h6A) was cloned from a human kidney cDNA library in 2004 (1). Cloning and deorphanization of the mouse (2, 3) and rat (4) GPRC6A orthologs rapidly followed. These studies, together with recent evidence (5) support the existence of GPRC6A as a dimer on the cell surface. However, surprisingly, h6A has been shown to be retained intracellularly and thus does not respond to agonists, which contrasts findings for the mouse, rat, and goldfish orthologs (2, 3, 6). To deorphanize h6A, we generated chimeras between the human and goldfish GPRC6A orthologs. We found that a fusion of the human large extracellular amino-terminal domain (ATD) to the 7-transmembrane (7TM) and C-terminal domains of the orthologous goldfish 5.24 receptor allowed efficient surface expression of the chimera and thereby a way to

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

“…To minimize effects from omnipresent agonists, we chose to test the rat GPRC6A receptor in the current study as amino acids are less potent at the rat receptor compared with the human receptor. Also, the rat receptor displays higher surface expression compared with the mouse and human orthologs (1,7,46), which should result in a higher signal-to-noise response in both the antibody feeding and real-time internalization assays. Accordingly, we were unable to test the human receptor in the current experimental setups due to the lower expression level (data not shown).…”

Osteocalcin Induces Proliferation via Positive Activation of the PI3K/Akt, P38 MAPK Pathways and Promotes Differentiation Through Activation of the GPRC6A-ERK1/2 Pathway in C2C12 Myoblast Cells

“…The GPCR class C group 6 subtype A (GPRC6A) protein is a member of a family of G protein-coupled receptors that are sensitive to pertussis toxin [27]. GPRC6A, as the putative receptor for osteocalcin, is expressed by a wide variety of cell types, including pancreas, testes and other tissues that respond to circulating osteocalcin [28,29].…”

A multilayered post-GWAS assessment on genetic susceptibility to pancreatic cancer

“…384 GPRC6A-rs6907580 is a well-characterized stop-gain variant in exon 1 of GPRC6A (G 392 protein-coupled receptor family C group 6 member A). GPRC6A is expressed in 393 pancreatic β-cells and participates in endocrine metabolism 39 and this SNP is linked to a 394 non-functional variant of GPRC6A receptor protein 40 . Furthermore, LMI identified 395 rs17078438 (6q22.1) in RFX6, a pancreas-specific gene involved in endocrine 396 development 41 (Figure 5).…”

“…Honolulu, HI, US. 109 (40) University of Texas MD Anderson Cancer Center, Houston, TX, US. 110 (41) Population Health Department, QIMR Berghofer Medical Research 111 Institute, Brisbane, Queensland, Australia.…”