Pharmacology and physiological function of the orphan GPRC6A receptor.

The G protein-coupled receptor GPRC6A (GPCR, Class C, group 6, subtype A) is a Gq/11 -coupled receptor widely expressed in human and rodent tissues. The proposed endogenous ligands are L-amino acids, divalent cations, osteocalcin and testosterone. This MiniReview provides an updated overview of the literature including the latest in vitro and in vivo studies. GPRC6A forms homodimers, it undergoes constitutive internalization, and very interestingly, the reason for the intracellular retention of the human receptor has been revealed. Multiple physiological functions of GPRC6A have been suggested based on studies using three different global GPRC6A knockout (KO) mouse models where exon II, exon VI or the full locus has been deleted. The newest studies on the full locus GPRC6A KO model show intact glucose and bone homoeostasis with a minor reduction in serum osteocalcin levels. Unfortunately, the physiological function of the receptor remains elusive due to a general lack of consensus/validation of reported phenotypes of the different KO models, and more research is thus warranted to uncover the physiological function. Recent discoveries of human genetic variants that cause either a premature stop codon or an intracellular retention of the receptor point towards human population studies as the preferred approach to continue studies on the function of GPRC6A.

Metabolic and skeletal homeostasis are maintained in full locus GPRC6A knockout mice.

The G protein-coupled receptor class C, group 6, subtype A (GPRC6A) is suggested to have a physiological function in glucose and bone metabolism, although the precise role lacks consensus due to varying findings in different knockout (KO) mouse models and inconsistent findings on the role of osteocalcin, a proposed GPRC6A agonist. We have further characterized a full locus GPRC6A KO model with respect to energy metabolism, including a long-term high-dose glucocorticoid metabolic challenge. Additionally, we analyzed the microarchitecture of tibiae from young, middle-aged and aged GPRC6A KO mice and wildtype (WT) littermates. Compared to WT, vehicle-treated KO mice presented with normal body composition, unaltered insulin sensitivity and basal serum insulin and glucose levels. Corticosterone (CS) treatment resulted in insulin resistance, abnormal fat accrual, loss of lean mass and suppression of serum osteocalcin levels in both genotypes. Interestingly, serum osteocalcin and skeletal osteocalcin mRNA levels were significantly lower in vehicle-treated GPRC6A KO mice compared to WT animals. However, WT and KO age groups did not differ in long bone mass and structure assessed by micro-computed tomography. We conclude that GPRC6A is not involved in glucose metabolism under normal physiological conditions, nor does it mediate glucocorticoid-induced dysmetabolism in mice. Moreover, GPRC6A does not appear to possess a direct, non-compensable role in long bone microarchitecture under standard conditions.

Label-free dynamic mass redistribution analysis of endogenous adrenergic receptor signaling in primary preadipocytes and differentiated adipocytes.

INTRODUCTION: Adipose tissues release adipokines, which regulate energy intake and expenditure. G protein-coupled receptors (GPCRs) and associated signaling pathways in adipocytes are potentially important drug targets for conditions with disturbed energy metabolism. METHODS: The aim of the current study was to compare signaling of endogenously expressed GPCRs between primary preadipocytes and differentiated adipocytes using a novel state-of-the-art unbiased method that measures dynamic mass redistribution (DMR) in real-time. Adrenergic agonists were chosen since they control adipocyte functions such as lipolysis and glycogenolysis. RESULTS: Isoprenaline (ISO) and phenylephrine (PE) elicited concentration-dependent responses in preadipocytes and differentiated adipocytes. The effect of ISO was cholera toxin (CTX)-sensitive, indicating it is Gs-dependent. The effect could also be blocked by propranolol proving the signal is mediated through ß-adrenergic receptors. The signaling resulting from PE stimulation was completely abolished by the Gq/11-selective inhibitor FR900359 and CTX in preadipocytes but surprisingly became FR900359-insensitive but remained CTX-sensitive in differentiated adipocytes. The use of prazosin and propranolol revealed that the PE-response in differentiated adipocytes had a ß-adrenergic receptor component to it. In addition, we tested the bone-derived peptide osteocalcin, which did not result in DMR changes in preadipocytes or differentiated adipocytes. DISCUSSION: In conclusion, this study for the first time demonstrates that DMR assays can be used to assess signaling in differentiated adipocytes. This platform can serve as a tool for future drug screening in primary adipocytes. Furthermore, this study illustrates that PE-induced effects on adipocytes vary by developmental stage and are not as selective as originally thought.

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

GPRC6A is a G protein-coupled receptor activated by l-amino 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 Gq-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.

Robust GLP-1 secretion by basic L-amino acids does not require the GPRC6A receptor.

The G protein-coupled receptor GPRC6A (GPCR, Class C, group 6, subtype A) has been proposed to be a sensor for basic L-amino acids that are hypothesized to translate ingestive behaviour to endocrine information. However, the contribution of the GPRC6A receptor to L-amino acid-induced glucagon-like peptide 1 (GLP-1) secretion is unclear. Therefore, to discover whether the GPRC6A receptor is indispensible for amino acid-induced secretion of GLP-1, we treated, with oral gavage, GPRC6A knock-out (KO) and wild-type (WT) littermate mice with GPRC6A ligands (L-arginine and L-ornithine) and assessed GLP-1 levels in circulation. We found that oral administration of both L-arginine and L-ornithine significantly increased total plasma GLP-1 levels to a similar level in GPRC6A KO and WT mice 15 minutes after gavage (both amino acids) and accumulated up to 60 minutes after gavage (L-arginine). Conversely, GLP-1 secretion at the 30- and 60-minute time points in the KO mice was attenuated and did not reach statistical significance. In summary, these data confirm that L-arginine is a potent GLP-1 secretagogue and show that the main effect occurs independently of GPRC6A. In addition, this is the first study to show that also L-ornithine powerfully elicits GLP-1 release in vivo.

Elevated levels of kynurenic acid during gestation produce neurochemical, morphological, and cognitive deficits in adulthood: implications for schizophrenia.

The levels of kynurenic acid (KYNA), an endogenous negative modulator of alpha7 nicotinic acetylcholine receptors (α7nAChRs), are elevated in the brains of patients with schizophrenia (SZ). We reported that increases of brain KYNA in rats, through dietary exposure to its precursor kynurenine from embryonic day (ED)15 to postnatal day (PD) 21, result in neurochemical and cognitive deficits in adulthood. The present experiments focused on the effects of prenatal exposure to elevated kynurenine on measures of prefrontal excitability known to be impaired in SZ. Pregnant dams were fed a mash containing kynurenine (100 mg/day; progeny = EKYNs) from ED15 until ED22. Controls were fed an unadulterated mash (progeny = ECONs). The dietary loading procedure elevated maternal and fetal plasma kynurenine (2223% and 693% above controls, respectively) and increased fetal KYNA (forebrain; 500% above controls) on ED21. Elevations in forebrain KYNA disappeared after termination of the loading (PD2), but KYNA levels in the prefrontal cortex (PFC) were unexpectedly increased again when measured in adults (PD56-80; 75% above controls). We also observed changes in several markers of prefrontal excitability, including expression of the α7nAChR (22% and 17% reductions at PD2 and PD56-80), expression of mGluR2 (31% and 24% reductions at ED21 and PD56-80), dendritic spine density (11-14% decrease at PD56-80), subsensitive mesolimbic stimulation of glutamate release in PFC, and reversal/extra-dimensional shift deficits in the prefrontally-mediated set-shifting task. These results highlight the deleterious impact of elevated KYNA levels during sensitive periods of early development, which model the pathophysiological and cognitive deficits seen in SZ.

Cerebral 5-HT2A receptor binding, but not mGluR2, is increased in tryptophan hydroxylase 2 decrease-of-function mice.

Transgenic mice with a knock-in (KI) of a tryptophan hydroxylase 2 (Tph2) R439H mutation, analogous to the Tph2 R441H single-nucleotide polymorphism originally identified in a late life depression cohort, have markedly reduced levels of 5-hydroxytryptamine (5-HT). These Tph2KI mice are therefore interesting as a putative translational model of low endogenous 5-HT function that allows for assessment of adaptive changes in different anatomical regions. Here, we determined 5-HT2A receptor binding in several brain regions using in vitro receptor autoradiography and two different radioligands. When using the 5-HT2A receptor selective antagonist radioligand (3)H-MDL100907, we found higher binding in the prefrontal cortex (10%, P=0.009), the striatum (26%, P=0.005), and the substantia nigra (21%, P=0.027). The increase was confirmed in the same regions with the 5-HT2A/C receptor agonist, (3)H-CIMBI-36 (2-(4-Bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine). 5-HT2A receptors establish heteromeric receptor complexes with metabotropic glutamate 2 receptors (mGluR2), but binding levels of the mGluR2/3 ligand (3)H-LY341495 were unaltered in brain areas with increased 5-HT2A receptor levels. These data show that in distinct anatomical regions, 5-HT2A receptor binding sites are up-regulated in 5-HT deficient mice, and this increase is not associated with changes in mGluR2 binding.

Metabotropic glutamate receptor 2 and corticotrophin-releasing factor receptor-1 gene expression is differently regulated by BDNF in rat primary cortical neurons.

Brain-derived neurotrophic factor (BDNF) is important for neuronal survival and plasticity. Incorporation of matured receptor proteins is an integral part of synapse formation. However, whether BDNF increases synthesis and integration of receptors in functional synapses directly is unclear. We are particularly interested in the regulation of the 5-hydroxytryptamine receptor 2A (5-HT(2A)R). This receptor form a functional complex with the metabotropic glutamate receptor 2 (mGluR2) and is recruited to the cell membrane by the corticotrophin-releasing factor receptor 1 (CRF-R1). The effect of BDNF on gene expression for all these receptors, as well as a number of immediate-early genes, was pharmacologically characterized in primary neurons from rat frontal cortex. BDNF increased CRF-R1 mRNA levels up to fivefold, whereas mGluR2 mRNA levels were proportionally downregulated. No effect on 5-HT(2A)R mRNA was seen. The effects were dose-dependent with half-maximal effective concentrations (EC(50)) around 1 ng/ml. After 24 h of incubation with BDNF, CRF-R1 mRNA levels had returned to baseline levels, whereas mGluR2 mRNA levels remained low. A significant reduction of all three receptor transcripts was observed after neuronal depolarization produced by high potassium. This study emphasizes the role of BDNF as an important regulator of receptor compositions in the synapse and provides further evidence that BDNF directly regulates important drug targets involved in cognition and mood.