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

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.

Synthesis and pharmacological evaluation of N-benzyl substituted 4-bromo-2,5-dimethoxyphenethylamines as 5-HT2A/2C partial agonists.

N-Benzyl substitution of phenethylamine 5-HT2A receptor agonists has dramatic effects on binding affinity, receptor selectivity and agonist activity. In this paper we examine how affinity for the 5-HT2A/2C receptors are influenced by N-benzyl substitution of 4-bromo-2,5-dimethoxyphenethylamine derivatives. Special attention is given to the 2' and 3'-position of the N-benzyl as such compounds are known to be very potent. We found that substitutions in these positions are generally well tolerated. The 2'-position was further examined using a range of substituents to probe the hydrogen bonding requirements for optimal affinity and selectivity, and it was found that small changes in the ligands in this area had a profound effect on their affinities. Furthermore, two ligands that lack a 2'-benzyl substituent were also found to have high affinity contradicting previous held notions. Several high-affinity ligands were identified and assayed for functional activity at the 5-HT2A and 5-HT2C receptor, and they were generally found to be less efficacious agonists than previously reported N-benzyl phenethylamines.

Delineation of the GPRC6A receptor signaling pathways using a mammalian cell line stably expressing the receptor.

The GPRC6A receptor is a recently "deorphanized" class C G protein-coupled receptor. We and others have shown that this receptor is coactivated by basic l-α-amino acids and divalent cations, whereas other groups have also suggested osteocalcin and testosterone to be agonists. Likewise, the GPRC6A receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort to establish fully the signaling properties of the receptor, we tested representatives of four previously reported GPRC6A agonist classes for activity in the Gq, Gs, Gi, and extracellular-signal regulated kinase signaling pathways. Our results confirm that GPRC6A is activated by basic l-α-amino acids and divalent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A is thus useful in high-throughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor.

Strontium is a biased agonist of the calcium-sensing receptor in rat medullary thyroid carcinoma 6-23 cells.

The calcium-sensing receptor (CaSR)-specific allosteric modulator cinacalcet has revolutionized the treatment of secondary hyperparathyroidism in patients with chronic kidney disease. However, its application is limited to patients with end-stage renal disease because of hypocalcemic side effects presumably caused by CaSR-mediated calcitonin secretion from thyroid parafollicular C-cells. These hypocalcemic side effects might be dampened by compounds that bias the signaling of CaSR, causing similar therapeutic effects as cinacalcet without stimulating calcitonin secretion. Because biased signaling of CaSR is poorly understood, the objective of the present study was to investigate biased signaling of CaSR by using rat medullary thyroid carcinoma 6-23 cells as a model of thyroid parafollicular C-cells. By doing concentration-response experiments we focused on the ability of two well known CaSR agonists, calcium and strontium, to activate six different signaling entities: G(q/11) signaling, G(i/o) signaling, G(s) signaling, extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling, intracellular calcium ([Ca(2+)](i)) mobilization, and calcitonin secretion. The experiments showed that strontium biases CaSR signaling toward ERK1/2 signaling and possibly another pathway independent of G(q/11) signaling and [Ca(2+)](i) mobilization. It is noteworthy that the potency of strontium-stimulated calcitonin secretion was elevated compared with calcium. Combining these results with experiments investigating signaling pathway components involved in calcitonin secretion, we found that the enhanced potency of strontium-mediated calcitonin secretion was caused by a different signaling pattern than that produced by calcium. Together, our results suggest that calcitonin secretion can be affected by CaSR-stimulated signaling bias, which may be used to develop novel drugs for the treatment of secondary hyperparathyroidism.

Investigating the molecular mechanism of positive and negative allosteric modulators in the calcium-sensing receptor dimer.

Allosteric modulators that are targeting the calcium-sensing receptor (CaSR) hold great therapeutic potential, and elucidating the molecular basis for modulation would thus benefit the development of novel therapeutics. In the present study, we aimed at investigating the mechanism of allosteric modulation in CaSR by testing dimers carrying mutations in the allosteric site of one or both of the subunits. To ensure measurements on a well-defined dimer composition, we applied a trans-activation system in which only the specific heterodimer of two loss-of-function mutants responded to agonist. Although one of these mutants was potentiated by a positive allosteric modulator, we showed that receptor activity was further potentiated in a trans-activation heterodimer containing a single allosteric site, however only when the allosteric site was located in the subunit responsible for G protein coupling. On the contrary, preventing activation in both subunits was necessary for obtaining full inhibition by a negative allosteric modulator. These findings correlate with the proposed activation mechanism of the metabotropic glutamate receptors (mGluRs), in which only a single transmembrane domain is activated at a time. CaSR and mGluRs belong to the class C G protein-coupled receptors, and our findings thus suggest that the activation mechanism is common to this subfamily.