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Bi-allelic loss-of-function variants in JKAMP cause a neurodevelopmental syndrome associated with dysregulation of GPR37 trafficking.

The endoplasmic reticulum (ER) serves as a key hub for protein homeostasis, maintaining a strict quality-control system that ensures only properly folded proteins reach their destinations, while misfolded proteins are degraded via ER-associated degradation (ERAD) or selective ER-phagy. JKAMP, which encodes an ER-resident transmembrane protein involved in ERAD, has not previously been associated with human disease. Here, we report bi-allelic loss-of-function variants in JKAMP in 14 affected individuals from 10 unrelated families presenting with a neurodevelopmental syndrome characterized by intellectual disability, developmental delay, seizures, hypotonia, microcephaly, and dysmorphic features. An in vivo zebrafish model lacking jkamp recapitulated key aspects of the human disorder, including developmental abnormalities and impaired myelin production, further corroborating its pathogenic role. Mechanistic studies identified GPR37, a brain-enriched orphan G protein-coupled receptor (GPCR) and known JKAMP interactor, as a critical downstream effector. GPR37 plays essential roles in dopaminergic signaling, inflammatory pain regulation, neuroprotection, and myelination. Loss of JKAMP resulted in defective folding and degradation of GPR37, leading to its accumulation within the ER and impaired trafficking to the plasma membrane, likely due to impaired ER quality control. These findings establish JKAMP as a previously unrecognized contributor to human neurodevelopment and uncover a pathogenic mechanism linking ER protein quality control to GPCR regulation and neurological disease.

Humans↗

An ITIM-like motif within the CCK2 receptor sequence required for interaction with SHP-2 and the activation of the AKT pathway.

SHP-2 is a tyrosine phosphatase which functions as a positive regulator downstream of RTKs, activating growth-stimulatory signalling pathways. To date, very few G protein-coupled receptors (GPCRs) have been shown to be connected to SHP-2 and very little is known about the positive role of SHP-2 in GPCR signalling. The CCK2 receptor (CCK2R), a GPCR, is now recognized to mediate mitogenic effects of gastrin on gastrointestinal cells. In the present study, we demonstrate the role of SHP-2 in the activation of the AKT pathway by the CCK2R in COS-7 cells transfected with the CCK2R and in a pancreatic cancer cell line expressing the endogenous receptor. Using surface plasmon resonance analysis, we identified a highly conserved ITIM motif, containing the tyrosine residue 438, located in the C-terminal intracellular tail of the CCK2R which directly interacts with the SHP-2 SH2 domains. The interaction was confirmed by pull down assays and co-immunoprecipitation of the receptor with SHP-2. This interaction was transiently increased following gastrin stimulation of the CCK2R and correlated with the tyrosine phosphorylation of SHP-2. Mutational analysis of the key ITIM residue 438 confirmed that the CCK2R ITIM sequence is required for interaction with SHP-2 and the activation of the AKT pathway.

Amino Acid Motifs↗

Structural studies of the putative helix 8 in the human beta(2) adrenergic receptor: an NMR study.

The recently reported crystal structure of bovine rhodopsin revealed a cytoplasmic helix (helix 8) in addition to the seven transmembrane helices. This domain is roughly perpendicular to the transmembrane bundle in the presence of an interface and may be a loop-like structure in the absence of an interface. Several studies carried out on this domain suggested that it might act as a conformational switch between the inactive and activated states of this G-protein coupled receptor (GPCR). These results raised the question whether helix 8 may be an important feature of other GPCRs as well. To explore this question, we determined the structure of a peptide representing the putative helix 8 domain in another receptor that belongs to the rhodopsin family of GPCRs, the human beta(2) adrenergic receptor (hbeta(2)AR), using two-dimensional (1)H nuclear magnetic resonance (NMR). The key results from this structural study are that the putative helix 8 domain is helical in detergent and in DMSO while in water this region is disordered; the conformation is therefore dependent upon the environment. Comparison of data from five GPCRs suggests that these observations may be generally important for GPCR structure and function.

Amino Acid Sequence↗

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation.

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.

Adrenergic beta-Agonists↗

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val-d-Met-induced calcium influx.

Intracellular calcium (Ca(2+)) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca(2+) signaling. The addition of exogenous LPA induced dramatic Ca(2+) influx but not intracellular Ca(2+) release in U937 cells. LPA-induced Ca(2+) influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A(2) and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A(2)-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca(2+) influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca(2+) influx.

Arachidonic Acid↗

Molecular cloning and characterization of a novel Gq-coupled orphan receptor GPRg1 exclusively expressed in the central nervous system.

G-protein-coupled receptors (GPCRs) are important mediators of signal transduction and are therefore potential targets for pharmacological therapeutics. Here, we report the identification and characterization of an orphan GPCR, termed GPRg1, which was found in the GenBank database following searches with GPCR query sequences. Quantitative PCR analysis revealed that GPRg1 transcripts are expressed almost exclusively in the brain. Moreover, in situ hybridization experiments in brain demonstrated that GPRg1 is abundantly expressed in the ventrolateral region of caudate putamen, the habenular nucleus, the zona incerta, and the medial mammillary nucleus. In addition, overexpression of GPRg1 in 293-EBNA cells activates serum response factor mediated transcription, which was completely inhibited by the Gq/11 selective inhibitor YM-254890, indicating the coupling of GPRg1 with Gq/11. These findings suggest that GPRg1 is a candidate receptor for novel physiologically bioactive substrates and that it plays important roles in the central nervous system.

Amino Acid Sequence↗

Calmodulin interaction with peptides from G-protein coupled receptors measured with S-Tag labeling.

We have developed a quantitative assay of calmodulin (CaM) binding to S-Tag labeled peptides derived from G-protein coupled receptor (GPCR) sequences. CaM binding of peptides derived from the third intracellular loop (i3) of mu opioid receptor (MOR) was confirmed and the CaM-binding motif refined. A MORi3 peptide with a Lys > Ala substitution--shown to reduce CaM-binding of intact MOR--bound fivefold less avidly than the wild-type peptide. Screening peptides derived from i3 loops of other GPCR families confirmed 5HT1A, and identified muscarinic receptor 3, and melanocortin receptor 1, as proteins carrying CaM-binding domains. The use of S-Tag labeling can serve for rapid screening of putative CaM-binding domains in GPCRs.

Binding Sites↗

Heterodimerization and surface localization of G protein coupled receptors.

G protein coupled receptors (GPCRs) are one of the largest human gene families, and are targets for many important therapeutic drugs. Over the last few years, there has been a major paradigm shift in our understanding of how these receptors function. Formerly, GPCRs were thought to exist as monomers that, upon agonist occupation, activated a heterotrimeric G protein to alter the concentrations of specific second messengers. Until recently, this relatively linear cascade has been the standard paradigm for signaling by these molecules. However, it is now clear that this model is not adequate to explain many aspects of GPCR function. We now know that many, if not most, GPCRs form homo- and/or hetero-oligomeric complexes and interact directly with intracellular proteins in addition to G proteins. It now appears that many GPCRs may not function independently, but might more accurately be described as subunits of large multi-protein signaling complexes. These observations raise many important new questions; some of which include: (1) how many functionally and pharmacologically distinct receptor subtypes exist in vivo? (2) Which GPCRs physically associate, and in what stochiometries? (3) What are the roles of individual subunits in binding ligand and activating responses? (4) Are the pharmacological or signaling properties of GPCR heterodimers different from monomers? Since these receptors are the targets for a large number of clinically useful compounds, such information is likely to be of direct therapeutic importance, both in understanding how existing drugs work, but also in discovering novel compounds to treat disease.

Dimerization↗

Constitutive activity and inverse agonism at the alpha1adrenoceptors.

Mutations of G protein-coupled receptors (GPCR) can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The alpha(1B)adrenoceptor was the first GPCR in which point mutations were shown to trigger receptor activation. This article briefly summarizes some of the findings reported in the last several years on constitutive activity of the alpha(1)adrenoceptor subtypes, the location where mutations have been found in the receptors, the spontaneous activity of native receptors in recombinant as well as physiological systems. In addition, it will highlight how the analysis of the pharmacological and molecular properties of the constitutively active adrenoceptor mutants provided an important contribution to our understanding of the molecular mechanisms underlying the mechanism of receptor activation and inverse agonism.

Adrenergic alpha-1 Receptor Antagonists↗

Identification of CXCL13 as an agonist and CXCL11 as an inverse agonist for the viral G protein-coupled receptor ORF74.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent infection in humans, but under conditions of immune suppression, it may reactivate and contribute to severe diseases, including Kaposi's sarcoma (KS) and B-cell malignancies. The KSHV genome encodes a single G protein-coupled receptor (GPCR), open reading frame 74 (ORF74), which shows homology to human chemokine receptors. Since its identification in 1996, ORF74 has subsequently been shown to interact with a broad range of human CXC chemokines, as well as CCL1 and the viral chemokine vCCL2. Compared with many human chemokine receptors, ORF74 displays high basal activity. These properties allow ORF74 to deregulate host cellular pathways through constitutive and chemokine-modulated signaling. In this study, we evaluated several human chemokines that, to our knowledge, had not previously been tested in ORF74-dependent cellular assays. Whereas CXCL9, CXCL14, CXCL16 and CXCL17 did not interact with ORF74, CXCL13 was identified as an additional ORF74 agonist and CXCL11 as an inverse agonist. CXCL13 dose-dependently induced ORF74-mediated Ca2+ release, β-arrestin1/2 recruitment and chemotaxis, and enhanced basal nuclear factor κB (NF-κB) activity in ORF74-expressing cells. In contrast, CXCL11 showed no detectable ORF74 agonist activity in the calcium mobilization or chemotaxis assay, but antagonized CXCL1-induced responses in both readouts. CXCL11 also elicited inverse agonist-like responses in β-arrestin1/2 recruitment assays and reduced basal NF-κB signaling. Our study thus reveals CXCL13 and CXCL11 as two additional chemokine ligands for ORF74, further expanding the pharmacological profile of this viral GPCR.

Humans↗

Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans.

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.

Animals↗

Oral delivery of G protein-coupled receptor modulators: an explanation for the observed class difference.

G protein-coupled receptors (GPCRs) represent important targets for drug intervention. However, analysis of GPCR modulator drugs exhibits an important class difference, with many drugs available against aminergic GPCR targets, but relatively few against non-aminergic targets. The reason for this is that commonly drugs mimic the physicochemistry of the receptor ligand. Aminergic ligands generally exhibit physicochemical properties (molecular weight, lipophilicity and hydrogen bonding potential) that are consistent with extensive oral absorption. In contrast, non-aminergic ligands generally exhibit physicochemical properties that are at odds with oral delivery. Thus, combining required potency versus the receptor, with oral delivery potential is a significant challenge, and drug discovery becomes a question of finding the exceptional compound that lies at the edge of ADME space.

Administration, Oral↗

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells.

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca(2+) release and store-operated Ca(2+) entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1microM) and the Src inhibitor PP2 (10microM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca(2+) transients were reduced by imatinib and/or PP2. Ca(2+) transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca(2+) transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCalpha catalytic activity and PKCalpha co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca(2+) influx was reduced by complexing extracellular Ca(2+) with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca(2+) transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.

Animals↗

Cargo regulates clathrin-coated pit dynamics.

Clathrin-coated pits (CCPs) are generally considered a uniform population of endocytic machines containing mixed constitutive and regulated membrane cargo. Contrary to this view, we show that regulated endocytosis of G protein-coupled receptors (GPCRs) occurs preferentially through a subset of CCPs. Significantly, GPCR-containing CCPs are also functionally distinct, as their surface residence time is regulated locally by GPCR cargo via PDZ-dependent linkage to the actin cytoskeleton. Such cargo-regulated CCPs show delayed recruitment of dynamin and can undergo an abortive event in which clathrin coats separate from the plasma membrane without concomitant receptor endocytosis. Segregation of cargo into CCP subsets, combined with cargo-dependent control of CCP dynamics, suggests a simple kinetic mechanism to generate functional specialization early in the endocytic pathway and reduce competition between diverse endocytic cargo.

Actins↗

Cell signalling through thromboxane A2 receptors.

Thromboxane A2 receptors (TPs) are widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. Following the initial cloning of this receptor class from human placenta, the deduced amino acid sequence predicted seven-transmembrane spanning regions, four extracellular domains and four intracellular domains, making TP a member of the seven-transmembrane G-protein-coupled receptor (GPCR) super family. A single gene on chromosome 19p13.3 leads to the expression of two separate TP isoforms: TPalpha which is broadly expressed in numerous tissues, and a splice variant termed TPbeta which may have a more limited tissue distribution. Mutagenesis, photoaffinity labelling, and immunological studies have indicated that the ligand binding domains for this receptor may reside in both the transmembrane (TM) and extracellular regions of the receptor protein. In addition, separate studies have provided evidence that this receptor can couple to at least four separate G protein families. As a consequence, TP signalling has been shown to result in a broad range of cellular responses including phosphoinositide metabolism, calcium redistribution, cytoskeletal arrangement, integrin activation, kinase activation, and the subsequent nuclear signalling events involved in DNA synthesis, cell proliferation, cell survival and cell death. While activation of these different signalling cascades can all derive from TP stimulation, the relative signalling preference for a given cascade appears to be both tissue and cell specific. Finally, separate studies have indicated that TP signalling capacity can be both down-regulated by protein kinase activation and up-regulated by GPCR cross-signalling. Thus, the multitude of signalling events which derive from TP activation can themselves be modulated by endogenous cellular messengers.

Cells, Cultured↗

Chemokine receptor CCR5: insights into structure, function, and regulation.

CC chemokine receptor 5 (CCR5) is a seven-transmembrane, G protein-coupled receptor (GPCR) which regulates trafficking and effector functions of memory/effector T-lymphocytes, macrophages, and immature dendritic cells. It also serves as the main coreceptor for the entry of R5 strains of human immunodeficiency virus (HIV-1, HIV-2). Chemokine binding to CCR5 leads to cellular activation through pertussis toxin-sensitive heterotrimeric G proteins as well as G protein-independent signalling pathways. Like many other GPCR, CCR5 is regulated by agonist-dependent processes which involve G protein coupled receptor kinase (GRK)-dependent phosphorylation, beta-arrestin-mediated desensitization and internalization. This review discusses recent advances in the elucidation of the structure and function of CCR5, as well as the complex mechanisms that regulate CCR5 signalling and cell surface expression.

Animals↗

Bombesin and angiotensin II rapidly stimulate Src phosphorylation at Tyr-418 in fibroblasts and intestinal epithelial cells through a PP2-insensitive pathway.

Src is activated in response to a variety of growth factors and hormones that bind G protein-coupled receptors (GPCRs), and its activity is regulated by phosphorylation at key sites, including the autophosphorylation site Tyr-418 and the inhibitory site Tyr-529. To better understand the mechanisms controlling Src activation, we examined Src phosphorylation in Swiss 3T3 fibroblasts stimulated with bombesin and in IEC-18 intestinal epithelial cells stimulated with angiotensin II (Ang II). Phosphorylation at Src Tyr-418, the activation loop site, was rapidly and markedly increased after GPCR agonist addition in both cell types. However, treatment of intact cells with the selective Src family kinase inhibitor PP2, at concentrations which abolished Src-mediated phosphorylation of focal adhesion kinase (FAK) at Tyr-577, unexpectedly led to increased phosphorylation at Src Tyr-418 and diminished phosphorylation at Tyr-529. In Swiss 3T3 cells, PP2 enhanced Tyr-418 phosphorylation after 1 min of bombesin stimulation, while in IEC-18 cells, PP2 increased Ang II-stimulated Tyr-418 phosphorylation at all times tested. These results imply that a distinct, non-Src family kinase may be responsible for phosphorylating Src at Tyr-418 in intact fibroblasts and epithelial cells stimulated by GPCR agonists.

3T3 Cells↗

Pathophysiological roles of G-protein-coupled receptor kinases.

G-protein-coupled receptor kinases (GRKs) interact with the agonist-activated form of G-protein-coupled receptors (GPCRs) to effect receptor phosphorylation and to initiate profound impairment of receptor signalling, or desensitization. GPCRs form the largest family of cell surface receptors known and defects in GRK function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations. This review focuses on the physiological role of GRKs revealed by genetically modified animals but also develops the involvement of GRKs in human diseases as, Oguchi disease, heart failure, hypertension or rhumatoid arthritis. Furthermore, the regulation of GRK levels in opiate addiction, cancers, psychiatric diseases, cystic fibrosis and cardiac diseases is discussed. Both transgenic mice and human pathologies have demonstrated the importance of GRKs in the signalling pathways of rhodopsin, beta-adrenergic and dopamine-1 receptors. The modulation of GRK activity in animal models of cardiac diseases can be effective to restore cardiac function in heart failure and opens a novel therapeutic strategy in diseases with GPCR dysregulation.

Animals↗