CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

2000 ◽  
Vol 279 (6) ◽  
pp. C1986-C1992 ◽  
Author(s):  
Rammohan V. Rao ◽  
Eileen L. Holicky ◽  
Susan M. Kuntz ◽  
Laurence J. Miller
Keyword(s):  
G Protein ◽  
Chinese Hamster ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Ovary Cell ◽  
Guanine Nucleotide Binding Protein ◽  
Receptor Phosphorylation ◽  
Regulatory Domains ◽  
Ovary Cell Line ◽  
G Protein Coupled

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

scholarly journals GRKs as Modulators of Neurotransmitter Receptors

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 52
Author(s):  
Eugenia V. Gurevich ◽  
Vsevolod V. Gurevich
Keyword(s):  
G Protein ◽  
General Model ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Neurotransmitter Receptors ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Receptor Kinase ◽  
G Protein Coupled ◽  
Homologous Desensitization

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

scholarly journals Generating FSH antagonists and agonists through immunization against FSH receptor N-terminal decapeptides

1999 ◽  
Vol 22 (2) ◽  
pp. 151-159 ◽  
Author(s):  
L Abdennebi ◽  
L Couture ◽  
D Grebert ◽  
E Pajot ◽  
R Salesse ◽  
...  
Keyword(s):  
Chinese Hamster ◽  
Biochemical Properties ◽  
Ovary Cell ◽  
Fsh Receptor ◽  
Female Mice ◽  
Specific Receptors ◽  
Ovary Cell Line ◽  
G Protein Coupled ◽  
Follicle Maturation ◽  
Receptor Family

Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice.

scholarly journals Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization

Endocrinology ◽  
2007 ◽  
Vol 149 (3) ◽  
pp. 1415-1422 ◽  
Author(s):  
Adam J. Pawson ◽  
Elena Faccenda ◽  
Stuart Maudsley ◽  
Zhi-Liang Lu ◽  
Zvi Naor ◽  
...  
Keyword(s):  
G Protein ◽  
Regulatory Elements ◽  
Gnrh Receptor ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Type I ◽  
Receptor Desensitization ◽  
Gnrh Receptors ◽  
Carboxyl Terminal ◽  
G Protein Coupled

Regulatory elements present in the cytoplasmic carboxyl-terminal tails of G protein-coupled receptors contribute to agonist-dependent receptor desensitization, internalization, and association with accessory proteins such as β-arrestin. The mammalian type I GnRH receptors are unique among the rhodopsin-like G protein-coupled receptors because they lack a cytoplasmic carboxyl-terminal tail. In addition, they do not recruit β-arrestin, nor do they undergo rapid desensitization. By measuring the internalization of labeled GnRH agonists, previous studies have reported that mammalian type I GnRH receptors undergo slow agonist-dependent internalization. In the present study, we have measured the internalization of epitope-tagged GnRH receptors, both in the absence and presence of GnRH stimulation. We demonstrate that mammalian type I GnRH receptors exhibit a low level of constitutive agonist-independent internalization. Stimulation with GnRH agonist did not significantly enhance the level of receptor internalization above the constitutive level. In contrast, the catfish GnRH and rat TRH receptors, which have cytoplasmic carboxyl-terminal tails, displayed similar levels of constitutive agonist-independent internalization but underwent robust agonist-dependent internalization, as did chimeras of the mammalian type I GnRH receptor with the cytoplasmic carboxyl-terminal tails of the catfish GnRH receptor or the rat TRH receptor. When the carboxyl-terminal Tyr325 and Leu328 residues of the mammalian type I GnRH receptor were replaced with alanines, these two mutant receptors underwent significantly impaired internalization, suggesting a function for the Tyr-X-X-Leu sequence in mediating the constitutive agonist-independent internalization of mammalian type I GnRH receptors. These findings provide further support for the underlying notion that the absence of the cytoplasmic carboxyl-terminal tail of the mammalian type I GnRH receptors has been selected for during evolution to prevent rapid receptor desensitization and internalization to allow protracted GnRH signaling in mammals.

scholarly journals Clathrin and GRK2/3 inhibitors block δ-opioid receptor internalization in myenteric neurons and inhibit neuromuscular transmission in the mouse colon

2019 ◽  
Vol 317 (2) ◽  
pp. G79-G89 ◽  
Author(s):  
Jesse J. DiCello ◽  
Pradeep Rajasekhar ◽  
Emily M. Eriksson ◽  
Ayame Saito ◽  
Arisbel B. Gondin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Opioid Receptor ◽  
Neuromuscular Transmission ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Enteric Neurons ◽  
Myenteric Neurons ◽  
Gut Function ◽  
G Protein Coupled

Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.

scholarly journals Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell.

1995 ◽  
Vol 130 (3) ◽  
pp. 579-590 ◽  
Author(s):  
B F Roettger ◽  
R U Rentsch ◽  
E M Hadac ◽  
E H Hellen ◽  
T P Burghardt ◽  
...  
Keyword(s):  
G Protein ◽  
Acinar Cell ◽  
Chinese Hamster ◽  
Pancreatic Acinar Cell ◽  
Endocytic Pathway ◽  
Ovary Cell ◽  
Dependent Manner ◽  
Lateral Mobility ◽  
Ultrastructural Studies ◽  
G Protein Coupled

Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell could be particularly effective to protect the cell from processes which might initiate pancreatitis, while providing for the rapid resensitization of this receptor to ensure appropriate pancreatic secretion to aid in nutrient assimilation for the organism.

scholarly journals Methods for Studying Endocytotic Pathways of Herpesvirus Encoded G Protein-Coupled Receptors

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5710
Author(s):  
Maša Mavri ◽  
Katja Spiess ◽  
Mette Marie Rosenkilde ◽  
Catrin Sian Rutland ◽  
Milka Vrecl ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Drug Targets ◽  
Current Knowledge ◽  
Immune Surveillance ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Linear Motif ◽  
Infection Processes ◽  
G Protein Coupled

Endocytosis is a fundamental process involved in trafficking of various extracellular and transmembrane molecules from the cell surface to its interior. This enables cells to communicate and respond to external environments, maintain cellular homeostasis, and transduce signals. G protein-coupled receptors (GPCRs) constitute a family of receptors with seven transmembrane alpha-helical domains (7TM receptors) expressed at the cell surface, where they regulate physiological and pathological cellular processes. Several herpesviruses encode receptors (vGPCRs) which benefits the virus by avoiding host immune surveillance, supporting viral dissemination, and thereby establishing widespread and lifelong infection, processes where receptor signaling and/or endocytosis seem central. vGPCRs are rising as potential drug targets as exemplified by the cytomegalovirus-encoded receptor US28, where its constitutive internalization has been exploited for selective drug delivery in virus infected cells. Therefore, studying GPCR trafficking is of great importance. This review provides an overview of the current knowledge of endocytic and cell localization properties of vGPCRs and methodological approaches used for studying receptor internalization. Using such novel approaches, we show constitutive internalization of the BILF1 receptor from human and porcine γ-1 herpesviruses and present motifs from the eukaryotic linear motif (ELM) resources with importance for vGPCR endocytosis.

scholarly journals A High-Throughput Small-Molecule Ligand Screen Targeted to Agonists and Antagonists of the G-Protein-Coupled Receptor GPR54

2010 ◽  
Vol 15 (5) ◽  
pp. 508-517 ◽  
Author(s):  
Wendy Kuohung ◽  
Maria Burnett ◽  
Deepa Mukhtyar ◽  
Eli Schuman ◽  
Jake Ni ◽  
...  
Keyword(s):  
G Protein ◽  
Small Molecule ◽  
High Throughput ◽  
Inositol Phosphate ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Functional Assay ◽  
G Protein Coupled Receptor ◽  
Steroid Dependent ◽  
G Protein Coupled

Recent data have shown that the G-protein-coupled receptor GPR54 (also known as KiSS-1 receptor) regulates GnRH release from the hypothalamus. This essential role of GPR54 in controlling the hypothalamic-pituitary-gonadal axis makes it an attractive target for therapeutic intervention in reproductive and cancer medicine. Currently, there are no small-molecule modulators of GPR54 function for experimental or clinical use. To identify small-molecule compounds that modify GPR54 signal transduction, the authors have adapted a cell-based functional assay for high-throughput screening (HTS) using a commercially available homogeneous time-resolved fluorescence assay for inositol phosphate accumulation. They generated stable Chinese hamster ovary cell transfectants that express human GPR54 for use in this assay. After optimization in an automated HTS environment, they screened a library of 110,000 small-molecule compounds using 2 protocols, one to identify agonists and one to identify antagonists. Hits obtained in the primary screen were confirmed to be active in secondary in vitro assays. Compounds identified as agonists or antagonists from HTS and secondary screening will be characterized to identify agents with the potential to be developed as novel orally active agents to treat hormone-dependent disorders such as abnormal puberty, infertility, endometriosis, and sex steroid-dependent tumors.

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