ß-Arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor.

The vasopressin type 2 receptor (V2R) is an essential G protein-coupled receptor (GPCR) in renal regulation of water homeostasis. Upon stimulation, the V2R activates Gαs and Gαq/11, which is followed by robust recruitment of ß-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the ß-arrestin association with the V2R does not terminate Gαs activation, and thus, Gαs-mediated signaling is sustained while the receptor is internalized. Here, we demonstrate that this V2R ability to co-interact with G protein/ß-arrestin and promote endosomal G protein signaling is not restricted to Gαs, but also involves Gαq/11. Furthermore, our data imply that ß-arrestins potentiate Gαs/Gαq/11 activation at endosomes rather than terminating their signaling. Surprisingly, we found that the V2R internalizes and promote endosomal G protein activation independent of ß-arrestins to a minor degree. These new observations challenge the current model of endosomal GPCR signaling and suggest that this event can occur in both ß-arrestin-dependent and -independent manners.

ß-arrestin-dependent and -independent endosomal G protein activation by the vasopressin type 2 receptor.

The vasopressin type 2 receptor (V2R) is an essential GPCR in renal regulation of water homeostasis. Upon stimulation, the V2R activates Gαs and Gαq/11, which is followed by robust recruitment of ß-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the ß-arrestin association with the V2R does not terminate Gαs activation, and thus, Gαs-mediated signaling is sustained while the receptor is internalized. Here, we demonstrate that this V2R ability to co-interact with G protein/ß-arrestin and promote endosomal G protein signaling is not restricted to Gαs, but also involves Gαq/11. Furthermore, our data implies that ß-arrestins potentiate Gαs/Gαq/11 activation at endosomes rather than terminating their signaling. Surprisingly, we found that the V2R internalizes and promote endosomal G protein activation independent of ß-arrestins to a minor degree. These new observations challenge the current model of endosomal GPCR signaling and suggest that this event can occur in both ß-arrestin-dependent and -independent manners.

G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology.

Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers D-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3',5'-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.

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.

Novel strategies in drug discovery of the calcium-sensing receptor based on biased signaling.

A hallmark of chronic kidney disease is hyperphosphatemia due to renal phosphate retention. Prolonged parathyroid gland exposure to hyperphosphatemia leads to secondary hyperparathyroidism characterized by hyperplasia of the glands and excessive secretion of parathyroid hormone (PTH), which causes renal osteodystrophy. PTH secretion from the parathyroid glands is controlled by the calcium-sensing receptor (CaSR) that senses extracellular calcium. High extracellular calcium activates the CaSR causing inhibition of PTH secretion through multiple signaling pathways. Cinacalcet is the first drug targeting the CaSR and can be used to effectively control and reduce PTH secretion in PTH-related diseases. Cinacalcet is a positive allosteric modulator of the CaSR and affects PTH secretion from parathyroid glands by shifting the calcium-PTH concentration-response curve to the left. One major disadvantage of cinacalcet is its hypocalcemic side effect, which may be caused by increased CaSR-mediated calcitonin secretion from the thyroid gland. However, multiple studies indicate that PTH and calcitonin secretion are stimulated by different signaling pathways, and therefore it might be possible to develop a CaSR activating drug that selectively activates signaling pathways that inhibit PTH secretion while having no effect on signaling pathways involved in calcitonin secretion. Such a drug would have the same therapeutic value as cinacalcet in lowering PTH secretion while eliminating the side effect of hypocalcemia by virtue of it not affecting calcitonin secretion. The present review will focus on recent advancements in understanding signaling and biased signaling of the CaSR, and how that may be utilized to discover new and smarter drugs targeting the CaSR.

Biased agonism of the calcium-sensing receptor.

After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off" conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G(q/11) protein, G(i/o) protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G(q/11) and G(i/o) signaling, ERK1/2 is activated through recruitment of ß-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.

Chemogenomic discovery of allosteric antagonists at the GPRC6A receptor.

GPRC6A is a Family C G protein-coupled receptor recently discovered and deorphanized by our group. This study integrates chemogenomic ligand inference, homology modeling, compound synthesis, and pharmacological mechanism-of-action studies to disclose two noticeable results of methodological and pharmacological character: (1) chemogenomic lead identification through the first, to our knowledge, ligand inference between two different GPCR families, Families A and C; and (2) the discovery of the most selective GPRC6A allosteric antagonists discovered to date. The unprecedented inference of pharmacological activity across GPCR families provides proof-of-concept for in silico approaches against Family C targets based on Family A templates, greatly expanding the prospects of successful drug design and discovery. The antagonists were tested against a panel of seven Family A and C G protein-coupled receptors containing the chemogenomic binding sequence motif where some of the identified GPRC6A antagonists showed some activity. However, three compounds with at least ∼3-fold selectivity for GPRC6A were discovered, which present a significant step forward compared with the previously published GPRC6A antagonists, calindol and NPS 2143, which both display ∼30-fold selectivity for the calcium-sensing receptor compared to GPRC6A. The antagonists constitute novel research tools toward investigating the signaling mechanism of the GPRC6A receptor at the cellular level and serve as initial ligands for further optimization of potency and selectivity enabling future ex vivo/in vivo pharmacological studies.

Diagnostic yield, interpretation, and clinical utility of mutation screening of sarcomere encoding genes in Danish hypertrophic cardiomyopathy patients and relatives.

The American Heart Association (AHA) recommends family screening for hypertrophic cardiomyopathy (HCM). We assessed the outcome of family screening combining clinical evaluation and screening for sarcomere gene mutations in a cohort of 90 Danish HCM patients and their close relatives, in all 451 persons. Index patients were screened for mutations in all coding regions of 10 sarcomere genes (MYH7, MYL3, MYBPC3, TNNI3, TNNT2, TPM1, ACTC, CSRP3, TCAP, and TNNC1) and five exons of TTN. Relatives were screened for presence of minor or major diagnostic criteria for HCM and tracking of DNA variants was performed. In total, 297 adult relatives (>18 years) (51.2%) fulfilled one or more criteria for HCM. A total of 38 HCM-causing mutations were detected in 32 index patients. Six patients carried two disease-associated mutations. Twenty-two mutations have only been identified in the present cohort. The genetic diagnostic yield was almost twice as high in familial HCM (53%) vs. HCM of sporadic or unclear inheritance (19%). The yield was highest in families with an additional history of HCM-related clinical events. In relatives, 29.9% of mutation carriers did not fulfil any clinical diagnostic criterion, and in 37.5% of relatives without a mutation, one or more criteria was fulfilled. A total of 60% of family members had no mutation and could be reassured and further follow-up ceased. Genetic diagnosis may be established in approximately 40% of families with the highest yield in familial HCM with clinical events. Mutation-screening was superior to clinical investigation in identification of individuals not at increased risk, where follow-up is redundant, but should be offered in all families with relatives at risk for developing HCM.