<i>N</i>‐glycosylation and disulfide bonding affects GPRC6A receptor expression, function, and dimerization

Nørskov-Lauritsen, Lenea; Jørgensen, Stine; Bräuner‐Osborne, Hans

doi:10.1016/j.febslet.2015.01.019

Cited by 31 publications

(37 citation statements)

References 61 publications

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“…In addition, we observe a significant boost of the h6A_KGRKLP response by co-transfection with G␣ q (G66D) (Fig. 3B) as we have previously done for m6A (5,9).…”

Section: Resultssupporting

confidence: 81%

“…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).…”

supporting

confidence: 72%

“…Total receptor expression was determined after permeabilization with the detergent Triton X-100 and used to calculate the ratio between surface and total receptor expression as shown in the figures (28). Because m6A, in our hands, signals via the G q pathway (11), we then tested the functional importance of ICL3 and the C-terminal tail in an IP-One assay, which measures the accumulation of D-myo-inositol monophosphate (IP 1 ) as a consequence of G q activation (5). We have previously shown that the co-transfection with G␣ q (G66D) boosts the m6A response (5, 9) without changing the pharmacological profile of the m6A receptor (10,11).…”

Section: Resultsmentioning

confidence: 99%

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

Jørgensen

Have

Underwood

et al. 2017

Journal of Biological Chemistry

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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

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“…In addition, we observe a significant boost of the h6A_KGRKLP response by co-transfection with G␣ q (G66D) (Fig. 3B) as we have previously done for m6A (5,9).…”

Section: Resultssupporting

confidence: 81%

supporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

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

Jørgensen

Have

Underwood

et al. 2017

Journal of Biological Chemistry

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“…It has been demonstrated that GPRC6A undergoes N-glycosylation (21), which is a posttranslational modification involved in maturation of receptors and correct surface targeting of mature proteins. Oligomerization is another important mechanism that controls surface targeting and/or function.…”

mentioning

confidence: 99%

“…Oligomerization is another important mechanism that controls surface targeting and/or function. For class C receptors in particular, homo-or heterodimerization has proven essential for receptor function, and accordingly homodimerization has been demonstrated for the GPRC6A receptor (2,21). However, the regula-tory mechanisms involved in termination of GPRC6A function remain unknown.…”

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confidence: 99%

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

Jacobsen

Ammendrup-Johnsen

Jansen

et al. 2017

Journal of Biological Chemistry

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The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure.The G protein-coupled receptor, class C, group 6, member A (GPRC6A), 2 is a nutrient-sensing G protein-coupled receptor (GPCR) belonging to class C. It is activated by basic L-␣-amino acids and divalent cations, which trigger coupling to the G q protein and thereby an increase in the intracellular levels of the second messengers inositol trisphosphate and Ca 2ϩ (1-8). Other ligands and signaling pathways have been reported (3, 9 -13); however Rueda et al. (8) and we (7) have not been able to confirm these findings. The GPRC6A receptor displays a broad but low expression profile in human, mouse, and rat (2,5,10,11,14,15), thus giving little indication to the physiological role of the receptor. Several groups have conducted studies using GPRC6A knock-out mice to elucidate the physiological function of the receptor, and although results differ between knockout mouse models, they altogether suggest an involvement in metabolism and endocrine regulation (6, 10, 11, 16 -18).Over the years, it has become evident that GPCR signaling is much more complex than once believed. For most receptors, a variety of ligands and intracellular signaling pathways are available, and numerous regulatory mechanisms are thus required for obtaining specific biological responses (19).

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Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction

Goolam

Brown

Edwards

et al. 2020

Journal of Bone and Mineral Research

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The calcium-sensing receptor is a homodimeric class C G protein-coupled receptor (GPCR) that senses extracellular Ca 2+ (Ca 2+ o ) via a dimeric extracellular Venus flytrap (VFT) unit that activates G protein-dependent signaling via twin Cysteine-rich domains linked to transmembrane heptahelical (HH) bundles. It plays a key role in the regulation of human calcium and thus mineral metabolism. However, the nature of interactions between VFT units and HH bundles, and the impacts of heterozygous or homozygous inactivating mutations, which have implications for disorders of calcium metabolism are not yet clearly defined. Herein we generated CaSR-GABA B1 and CaSR-GABA B2 chimeras subject to GABA B -dependent endoplasmic reticulum sorting to traffic mutant heterodimers to the cell surface. Transfected HEK-293 cells were assessed for Ca 2+ o -stimulated Ca 2+ i mobilization using mutations in either the VFT domains and/or HH bundle intraloop-2 or intraloop-3. When the same mutation was present in both VFT domains of receptor dimers, analogous to homozygous neonatal severe hyperparathyroidism (NSHPT), receptor function was markedly impaired. Mutant heterodimers containing one wild-type (WT) and one mutant VFT domain, however, corresponding to heterozygous familial hypocalciuric hypercalcemia type-1 (FHH-1), supported maximal signaling with reduced Ca 2+ o potency. Thus two WT VFT domains were required for normal Ca 2+ o potency and there was a pronounced gene-dosage effect. In contrast, a single WT HH bundle was insufficient for maximal signaling and there was no functional difference between heterodimers in which the mutation was present in one or both intraloops; ie, no gene-dosage effect. Finally, we observed that the Ca 2+ o -stimulated CaSR operated exclusively via signaling in-trans and not via combined in-trans and in-cis signaling. We consider how receptor asymmetry may support the underlying mechanisms.

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N‐glycosylation and disulfide bonding affects GPRC6A receptor expression, function, and dimerization

Cited by 31 publications

References 61 publications

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

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

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

Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction

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