Differential G protein activation by the long and short isoforms of the dopamine D2 receptor.

BACKGROUND AND PURPOSE: The dopamine D2 receptor is expressed as a short (D2S) and a long (D2L) isoform with 29 additional amino acids in the third intracellular loop. The D2S isoform shows higher presynaptic expression than the D2L isoform, and decreased D2S expression has recently been linked to an increased risk for schizophrenia. Here, we present the first investigation, at receptor isoform level, of kinetic differences in the G protein activation profiles of the D2S, compared with the D2L isoform. EXPERIMENTAL APPROACH: We employed a NanoBRET-based approach to G protein dissociation to interrogate the time-resolved coupling profile of 3×HA-tagged D2L and D2S to Gαi/o/z proteins in vitro. KEY RESULTS: Using dopamine as a D2 receptor agonist, we observed a more pronounced activation of Gαo and Gαz than Gαi proteins by D2L compared with D2S. This differentiation was not observed for D2S, which activated Gαo and Gαz with lower efficacy than D2L. These signalling differences were preserved on second messenger level and were not due to differences in receptor expression. Expanding to a set of seven full and partial D2 receptor agonists showed these effects were not restricted to dopamine but rather a mutual, receptor-associated property. Contrasting this trend, we found that D2S activated G proteins faster than D2L upon full receptor activation. CONCLUSION AND IMPLICATIONS: The findings highlight that both D2L and D2S are mechanistically able to activate all non-visual Gαi/o proteins. Thereby, they add to previous reports about isoform-specificity to certain Gαi/o proteins observed in specific cell types.

Structure-Activity Relationship Studies and Optimization of 4-Hydroxypyridones as GPR84 Agonists.

GPR84 is a putative medium-chain fatty acid receptor that is implicated in regulation of inflammation and fibrogenesis. Studies have indicated that GPR84 agonists may have therapeutic potential in diseases such as Alzheimer's disease, atherosclerosis, and cancer, but there is a lack of quality tool compounds to explore this potential. The fatty acid analogue LY237 (4a) is the most potent GPR84 agonist disclosed to date but has unfavorable physicochemical properties. We here present a SAR study of 4a. Several highly potent agonists were identified with EC50 down to 28 pM, and with SAR generally in excellent agreement with structure-based modeling. Proper incorporation of rings and polar groups resulted in the identification of TUG-2099 (4s) and TUG-2208 (42a), both highly potent GPR84 agonists with lowered lipophilicity and good to excellent solubility, in vitro permeability, and microsomal stability, which will be valuable tools for exploring the pharmacology and therapeutic prospects of GPR84.

Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins.

G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.

The Concise Guide to PHARMACOLOGY 2023/24: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms.

GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into ß-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

Structure-activity relationship of GPR15L peptide analogues and investigation of their interaction with the GPR15 receptor.

The 57-mer full-length GPR15L(25-81) peptide has been identified as the principal endogenous agonist of the G protein-coupled receptor GPR15. Its main activity resides in the C-terminal 11-mer GPR15L(71-81), which has full efficacy but ~40-fold lower potency than the full-length peptide. Here, we systematically investigated the structure-activity relationship of GPR15L(71-81) by truncations/extensions, alanine-scanning, and N- and C-terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time-resolved Förster resonance energy transfer-based Gi cAMP functional assay. We show that the C-terminal α carboxyl group and the residues Leu78 , Pro75 , Val74 , and Trp72 are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71-81), C-terminally amidated GPR15L(71-81), and GPR15L(25-81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer-based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.

Pharmacological characterization of novel small molecule agonists and antagonists for the orphan receptor GPR139.

The orphan G protein-coupled receptor GPR139 is predominantly expressed in the central nervous system and has attracted considerable interest as a therapeutic target. However, the biological role of this receptor remains somewhat elusive, in part due to the lack of quality pharmacological tools to investigate GPR139 function. In an effort to understand GPR139 signaling and to identify improved compounds, in this study we performed virtual screening and analog searches, in combination with multiple pharmacological assays. We characterized GPR139-dependent signaling using previously published reference agonists in Ca2+ mobilization and inositol monophosphate accumulation assays, as well as a novel real-time GPR139 internalization assay. For the four reference agonists tested, the rank order of potency was conserved across signaling and internalization assays: JNJ-63533054 > Compound 1a ¼ Takeda > AC4 > DL43, consistent with previously reported values. We noted an increased efficacy of JNJ-63533054-mediated inositol monophosphate signaling and internalization, relative to Compound 1a. We then performed virtual screening for GPR139 agonist and antagonist compounds that were screened and validated in GPR139 functional assays. We identified four GPR139 agonists that were active in all assays, with similar or reduced potency relative to known compounds. Likewise, compound analogs selected based on GPR139 agonist and antagonist substructure searches behaved similarly to their parent compounds. Thus, we have characterized GPR139 signaling for multiple new ligands using G protein-dependent assays and a new real-time internalization assay. These data add to the GPR139 tool compound repertoire, which could be optimized in future medical chemistry campaigns.

Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization.

The internalization of G protein-coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.

Development of a medium throughput whole-cell microtiter plate Thr286 autophosphorylation assay for CaMKIIα using ELISA.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a multifunctional Ser/Thr kinase involved in several neuronal signaling pathways including synaptic plasticity. CaMKIIα autonomous activity is highly dependent on Thr286 autophosphorylation (pThr286), which is widely used as a readout for its enzymatic activity. To readily characterise compounds and potential drug candidates targeting CaMKIIα, a simple, generic cell-based assay for quantification of pThr286 levels is needed. In this study, we present a cell-based assay using an adapted ELISA as a suitable and higher throughput alternative to Western blotting. In this 96-well plate-based assay, we use whole HEK293T cells recombinantly expressing CaMKIIα and apply a phospho-specific antibody to detect pThr286 levels by chemiluminescence. In parallel, total CaMKIIα expression levels are detected by fluorescence using an Alexa488-conjugated anti-myc antibody targeting a C-terminal myc-tag. By multiplexing chemiluminescence and fluorescence, phosphorylation levels are normalised to CaMKIIα total expression within each well. The specificity of the assay was confirmed using a phosphodead mutant (T286A) of CaMKIIα. By applying Ca2+ or known CaMKIIα inhibitors (KN93, tatCN21 and AS100105) and obtaining concentration-response curves, we demonstrate high sensitivity and validity of the assay. Lastly, we demonstrate the versatility of the assay by determining autophosphorylation levels in CaMKIIα patient-related mutations, known to possess altered pThr286 responses (E109D, E183V and H282R). The established assay for CaMKIIα is a reproducible, easily implemented, and facile ELISA-based assay that allows for reliable quantification of pThr286 levels.

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells.

The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25-81) and a C-terminal peptide fragment GPR15L(71-81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3',5'-monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs , Gq/11 and G12/13 is unclear. Here, GPR15L(25-81) and GPR15L(71-81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1 , Gi2 , Gi3 , GoA , GoB and Gz ). The Emax and activation rates of Gi1, Gi2 , Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin.

Somatostatin venom analogs evolved by fish-hunting cone snails: From prey capture behavior to identifying drug leads.

Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.

Ligand-directed bias of G protein signaling at the dopamine D2 receptor.

G-protein-coupled receptors (GPCRs) represent the largest family of drug targets. Upon activation, GPCRs signal primarily via a diverse set of heterotrimeric G proteins. Most GPCRs can couple to several different G protein subtypes. However, how drugs act at GPCRs contributing to the selectivity of G protein recognition is poorly understood. Here, we examined the G protein selectivity profile of the dopamine D2 receptor (D2), a GPCR targeted by antipsychotic drugs. We show that D2 discriminates between six individual members of the Gi/o family, and its profile of functional selectivity is remarkably different across its ligands, which all engaged D2 with a distinct G protein coupling pattern. Using structural modeling, receptor mutagenesis, and pharmacological evaluation, we identified residues in the D2 binding pocket that shape these ligand-directed biases. We further provide pharmacogenomic evidence that natural variants in D2 differentially affect its G protein biases in response to different ligands.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Asymmetric activation of the calcium-sensing receptor homodimer.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor.

Mutational Landscape of the Proglucagon-Derived Peptides.

Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.

GLP-1 Val8: A Biased GLP-1R Agonist with Altered Binding Kinetics and Impaired Release of Pancreatic Hormones in Rats.

Biased ligands that selectively confer activity in one pathway over another are pharmacologically important because biased signaling may reduce on-target side effects and improve drug efficacy. Here, we describe an N-terminal modification in the incretin hormone glucagon-like peptide (GLP-1) that alters the signaling capabilities of the GLP-1 receptor (GLP-1R) by making it G protein biased over internalization but was originally designed to confer DPP-4 resistance and thereby prolong the half-life of GLP-1. Despite similar binding affinity, cAMP production, and calcium mobilization, substitution of a single amino acid (Ala8 to Val8) in the N-terminus of GLP-1(7-36)NH2 (GLP-1 Val8) severely impaired its ability to internalize GLP-1R compared to endogenous GLP-1. In-depth binding kinetics analyses revealed shorter residence time for GLP-1 Val8 as well as a slower observed association rate. Molecular dynamics (MD) displayed weaker and less interactions of GLP-1 Val8 with GLP-1R, as well as distinct conformational changes in the receptor compared to GLP-1. In vitro validation of the MD, by receptor alanine substitutions, confirmed stronger impairments of GLP-1 Val8-mediated signaling compared to GLP-1. In a perfused rat pancreas, acute stimulation with GLP-1 Val8 resulted in a lower insulin and somatostatin secretion compared to GLP-1. Our study illustrates that profound differences in molecular pharmacological properties, which are essential for the therapeutic targeting of the GLP-1 system, can be induced by subtle changes in the N-terminus of GLP-1. This information could facilitate the development of optimized GLP-1R agonists.

Positive Allosteric Modulators of Metabotropic Glutamate Receptor 5 as Tool Compounds to Study Signaling Bias.

Positive allosteric modulation of metabotropic glutamate subtype 5 (mGlu5) receptor has emerged as a potential new therapeutic strategy for the treatment of schizophrenia and cognitive impairments. However, positive allosteric modulator (PAM) agonist activity has been associated with adverse side effects, and neurotoxicity has also been observed for pure PAMs. The structural and pharmacological basis of therapeutic versus adverse mGlu5 PAM in vivo effects remains unknown. Thus, gaining insights into the signaling fingerprints, as well as the binding kinetics of structurally diverse mGlu5 PAMs, may help in the rational design of compounds with desired properties. We assessed the binding and signaling profiles of N-methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA), 3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide (CDPPB), and 1-[4-(4-chloro-2-fluoro-phenyl)piperazin-1-yl]-2-(4-pyridylmethoxy)ethenone [compound 2c, a close analog of 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone] in human embryonic kidney 293A cells stably expressing mGlu5 using Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and receptor internalization assays. Of the three allosteric ligands, only CDPPB had intrinsic agonist efficacy, and it also had the longest receptor residence time and highest affinity. MPPA was a biased PAM, showing higher positive cooperativity with orthosteric agonists in ERK1/2 phosphorylation and Ca2+ mobilization over IP1 accumulation and receptor internalization. In primary cortical neurons, all three PAMs showed stronger positive cooperativity with (S)-3,5-dihydroxyphenylglycine (DHPG) in Ca2+ mobilization over IP1 accumulation. Our characterization of three structurally diverse mGlu5 PAMs provides further molecular pharmacological insights and presents the first assessment of PAM-mediated mGlu5 internalization. SIGNIFICANCE STATEMENT: Enhancing metabotropic glutamate receptor subtype 5 (mGlu5) activity is a promising strategy to treat cognitive and positive symptoms in schizophrenia. It is increasingly evident that positive allosteric modulators (PAMs) of mGlu5 are not all equal in preclinical models; there remains a need to better understand the molecular pharmacological properties of mGlu5 PAMs. This study reports detailed characterization of the binding and functional pharmacological properties of mGlu5 PAMs and is the first study of the effects of mGlu5 PAMs on receptor internalization.

Arrestin-Dependent and -Independent Internalization of G Protein-Coupled Receptors: Methods, Mechanisms, and Implications on Cell Signaling.

Agonist-induced endocytosis is a key regulatory mechanism for controlling the responsiveness of the cell by changing the density of cell surface receptors. In addition to the role of endocytosis in signal termination, endocytosed G protein-coupled receptors (GPCRs) have been found to signal from intracellular compartments of the cell. Arrestins are generally believed to be the master regulators of GPCR endocytosis by binding to both phosphorylated receptors and adaptor protein 2 (AP-2) or clathrin, thus recruiting receptors to clathrin-coated pits to facilitate the internalization process. However, many other functions have been described for arrestins that do not relate to their role in terminating signaling. Additionally, there are now more than 30 examples of GPCRs that internalize independently of arrestins. Here we review the methods, pharmacological tools, and cellular backgrounds used to determine the role of arrestins in receptor internalization, highlighting their advantages and caveats. We also summarize key examples of arrestin-independent GPCR endocytosis in the literature and their suggested alternative endocytosis pathway (e.g., the caveolae-dependent and fast endophilin-mediated endocytosis pathways). Finally, we consider the possible function of arrestins recruited to GPCRs that are endocytosed independently of arrestins, including the catalytic arrestin activation paradigm. Technological improvements in recent years have advanced the field further, and, combined with the important implications of endocytosis on drug responses, this makes endocytosis an obvious parameter to include in molecular pharmacological characterization of ligand-GPCR interactions. SIGNIFICANCE STATEMENT: G protein-coupled receptor (GPCR) endocytosis is an important means to terminate receptor signaling, and arrestins play a central role in the widely accepted classical paradigm of GPCR endocytosis. In contrast to the canonical arrestin-mediated internalization, an increasing number of GPCRs are found to be endocytosed via alternate pathways, and the process appears more diverse than the previously defined "one pathway fits all."

The Calcium-Sensing Receptor Is Essential for Calcium and Bicarbonate Sensitivity in Human Spermatozoa.

CONTEXT: The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown. OBJECTIVE: This work aimed to investigate the role of CaSR in human spermatozoa. METHODS: We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and 3',5'-cyclic adenosine 5'-monophosphate (cAMP) in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors. RESULTS: CaSR is expressed in human spermatozoa and is essential for sensing extracellular free ionized calcium (Ca2+) and Mg2+. Activators of CaSR augmented the effect of sperm-activating signals such as the response to HCO3- and the acrosome reaction, whereas spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3-, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca2+ or Mg2+ was indispensable for HCO3- activation of sAC. Two male patients with a CASR loss-of-function mutation in exon 3 presented with normal sperm counts and motility, whereas a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology. CONCLUSION: CaSR is important for the sensing of Ca2+, Mg2+, and HCO3- in spermatozoa, and loss-of-function may impair male sperm function.