scholarly journals Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe

1999 ◽  
Vol 163 (3) ◽  
pp. 447-456 ◽  
Author(s):  
B Byrne ◽  
A McGregor ◽  
PL Taylor ◽  
R Sellar ◽  
FE Rodger ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Maximal Response ◽  
Wild Type ◽  
Releasing Hormone ◽  
Agonist And Antagonist ◽  
Gonadotrophin Releasing Hormone ◽  
Antagonist Action ◽  
G Protein Coupled ◽  
Type Receptor ◽  
D Value

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.

Characterization of the gonadotrophin-releasing hormone receptor in αT3-1 pituitary gonadotroph cells

1993 ◽  
Vol 136 (1) ◽  
pp. 51-NP ◽  
Author(s):  
L. Anderson ◽  
G. Milligan ◽  
K. A. Eidne
Keyword(s):  
G Protein ◽  
Inositol Phosphate ◽  
Rank Order ◽  
Second Messenger ◽  
Time Dependent ◽  
Binding Studies ◽  
Gnrh Analogues ◽  
Α Subunit ◽  
Releasing Hormone ◽  
Gonadotrophin Releasing Hormone

ABSTRACT The present study has characterized the gonadotrophin-releasing hormone (GnRH) receptor in immortalized αT3-1 pituitary gonadotroph cells. GnRH and GnRH analogues produced both a dose- and time-dependent increase in total inositol phosphate (IP) accumulation. The rank order of potency of these analogues was the same as that obtained in parallel receptor-binding studies in αT3-1 cells. These responses were abolished following pretreatment with a GnRH antagonist. The use of a specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) assay demonstrated a rapid but short-lived rise in Ins(1,4,5)P3 production. Intracellular calcium ([Ca2+]i) was subsequently measured in αT3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. GnRH produced a biphasic rise in [Ca2+]i. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. G-protein involvement in the IP response to GnRH in αT3-1 cells was investigated using sodium fluoride (NaF) and pertussis toxin (PTx) which activate and inactivate G-proteins respectively. Like GnRH, NaF produced a dose- and time-dependent increase in IP accumulation. Activation of phospholipase C in these cells by either GnRH or NaF was PTx-insensitive, suggesting that the G-protein involved was neither Gi nor Go but more probably Gq. Immunoblot analysis of αT3-1 cell membranes using antisera raised against the predicted C-terminal decapeptide of the α subunit of Gq demonstrated the presence of Gq in αT3-1 cells. Collectively these results show that the GnRH receptors expressed in αT3-1 cells are coupled to the phosphatidylinositol second messenger pathway via a specific G-protein. αT3-1 therefore represents a convenient model in which to study GnRH-related second messenger pathways. Journal of Endocrinology (1993) 136, 51–58

scholarly journals Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

1999 ◽  
Vol 274 (18) ◽  
pp. 12548-12554 ◽  
Author(s):  
Christian Le Gouill ◽  
Jean-Luc Parent ◽  
Carolyn-Ann Caron ◽  
Rémi Gaudreau ◽  
Léonid Volkov ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Selective Modulation ◽  
G Protein Coupled ◽  
Type Receptor

scholarly journals A constitutively active mutant of the α1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins Gqα and G11α than the wild-type receptor

1996 ◽  
Vol 320 (1) ◽  
pp. 79-86 ◽  
Author(s):  
Tae Weon LEE ◽  
Alan WISE ◽  
Susanna COTECCHIA ◽  
Graeme MILLIGAN
Keyword(s):  
G Proteins ◽  
Cell Lines ◽  
Adrenergic Receptor ◽  
Inositol Phosphate ◽  
Wild Type ◽  
Down Regulation ◽  
Eta Receptor ◽  
Type Receptor ◽  
In Cells ◽  
Constitutively Active

Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288–294 to encode the equivalent region of the human β2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAMα1B-adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAMα1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAMα1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the α subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins Gq and G11 in cells expressing either the wild-type or the CAMα1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAMα1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAMα1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of Gqα/G11α degradation between cells expressing the wild-type or the CAMα1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAMα1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAMα1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to Gq and G11.

Mutational analysis of the serotonin receptor 5HT2c in severe early-onset human obesity

2004 ◽  
Vol 82 (6) ◽  
pp. 426-429 ◽  
Author(s):  
William T Gibson ◽  
Barbara J Ebersole ◽  
Sumit Bhattacharyya ◽  
Peter Clayton ◽  
I Sadaf Farooqi ◽  
...  
Keyword(s):  
Early Onset ◽  
Serotonin Receptor ◽  
Mutational Analysis ◽  
Inositol Phosphate ◽  
Missense Variant ◽  
Human Obesity ◽  
Caucasian Subject ◽  
Cos Cells ◽  
G Protein Coupled ◽  
Type Receptor

Deletion of the serotonin receptor 5HT2c in mice results in increased food intake and obesity. We screened 95 individuals with severe early-onset obesity for mutations in the coding sequence of this gene. We found a novel missense variant c.1255A > G (Thr419Ala) in a single Caucasian subject that was not found in 192 Caucasian control subjects. In transiently-transfected COS cells, the Thr419Ala variant was indistinguishable from the wild-type receptor in its ability to generate inositol phosphate, although differences in coupling to other pathways were not excluded. Three previously unreported silent variants: IVS3 + 30G > A, IVS3 + 80C > G and IVS4 – 31A > G were found with prevalences of 11.5%, 0.5% and 17.9%, respectively. In conclusion, mutations in 5HT2c are unlikely to be a common cause of severe early-onset human obesity. The identification of several novel polymorphisms at this locus may aid future genetic epidemiological studies.Key words: G-protein coupled receptor, hyperphagia, obesity, serotonin, X-linked.

Gonadotrophin-releasing activity of neurohypophysial hormones: II. The pituitary oxytocin receptor mediating gonadotrophin release differs from that of corticotrophs

1989 ◽  
Vol 122 (1) ◽  
pp. 107-116 ◽  
Author(s):  
J. J. Evans ◽  
K. J. Catt
Keyword(s):  
Calcium Concentration ◽  
Phosphodiesterase Inhibitor ◽  
Pituitary Cells ◽  
Neurohypophysial Hormones ◽  
Gonadotrophin Secretion ◽  
Agonist And Antagonist ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone ◽  
Type Receptor

ABSTRACT Neurohypophysial hormones stimulate gonadotrophin release from dispersed rat anterior pituitary cells in vitro, acting through receptors distinct from those which mediate the secretory response to gonadotrophin-releasing hormone (GnRH). The LH response to oxytocin was not affected by the presence of the phosphodiesterase inhibitor, methyl isobutylxanthine, but was diminished in the absence of extracellular calcium and was progressively increased as the calcium concentration in the medium was raised to normal. In addition, the calcium channel antagonist, nifedipine, suppressed oxytocin-stimulated secretion of LH. It is likely that the mechanisms of LH release induced by GnRH and neurohypophysial hormones are similar, although stimulation of gonadotrophin secretion is mediated by separate receptor systems. Oxytocin was more active than vasopressin in releasing LH, but less active in releasing ACTH. The highly selective oxytocin agonist, [Thr4,Gly7]oxytocin, elicited concentration-dependent secretion of LH but had little effect on corticotrophin secretion. The neurohypophysial hormone antagonist analogues, [d(CH2)5Tyr(Me)2]-vasopressin, [d(CH2)5Tyr(Me)2,Orn8]vasotocin and [d(CH2)5d-Tyr(Et)2Val4,Cit8]vasopressin, inhibited the LH response to both oxytocin and vasopressin. However, [d(CH2)5Tyr(Me)2]vasopressin was much less effective in inhibiting the ACTH response to the neurohypophysial hormones, and [d(CH2)5Tyr-(Me)2,Orn8]vasotocin and [d(CH2)5d-Tyr(Et)2,Val4, Cit8]vasopressin exhibited no inhibitory activity against ACTH release. Thus, agonist and antagonist analogues of neurophypophysial hormones display divergent activities with regard to LH and ACTH responses, and the neuropeptide receptor mediating gonadotroph activation is clearly different from that on the corticotroph. Whereas the corticotroph receptor is a vasopressin-type receptor an oxytocin-type receptor is responsible for gonadotrophin release by neurohypophysial hormones. Journal of Endocrinology (1989) 122, 107–116

scholarly journals Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1

2007 ◽  
Vol 177 (5) ◽  
pp. 905-916 ◽  
Author(s):  
Breann L. Wolfe ◽  
Adriano Marchese ◽  
JoAnn Trejo
Keyword(s):  
G Protein ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Wild Type ◽  
Endocytic Machinery ◽  
Protease Activated Receptor 1 ◽  
Clathrin Adaptor ◽  
G Protein Coupled ◽  
Type Receptor

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

scholarly journals That was then, this is now: the development of our knowledge and understanding of P2 receptor subtypes

2021 ◽  
Author(s):  
Charles Kennedy
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Receptor Subtypes ◽  
Pyrimidine Nucleotides ◽  
Functional Studies ◽  
Agonist And Antagonist ◽  
Purine And Pyrimidine Nucleotides ◽  
Quaternary Structures ◽  
Antagonist Action ◽  
G Protein Coupled

AbstractP2 receptors are present in virtually all tissues and cell types in the human body, and they mediate the physiological and pharmacological actions of extracellular purine and pyrimidine nucleotides. They were first characterised and named by Geoff Burnstock in 1978, then subdivided into P2X and P2Y purinoceptors in 1985 on the basis of pharmacological criteria in functional studies on native receptors. Molecular cloning of receptors in the 1990s revealed P2X receptors to comprise seven different subunits that interact to produce functional homo- and heterotrimeric ligand-gated cation channels. A family of eight P2Y G protein–coupled receptors were also cloned, which can form homo- and heterodimers. Deep insight into the molecular mechanisms of agonist and antagonist action has been provided by more recent determination of the tertiary and quaternary structures of several P2X and P2Y receptor subtypes. Agonists and antagonists that are highly selective for individual subtypes are now available and some are in clinical use. This has all come about because of the intelligence, insight and drive of the force of nature that was Geoff Burnstock.

scholarly journals Binding and activation of serotonergic G-protein coupled receptors by the multimodal antidepressant vortioxetine

2021 ◽  
Author(s):  
Lucy Kate Ladefoged ◽  
Rebekka Koch ◽  
Philip C. Biggin ◽  
Birgit Schiøtt
Keyword(s):  
G Protein ◽  
Antidepressant Drug ◽  
G Protein Coupled Receptors ◽  
Differential Mode ◽  
Pharmacological Targets ◽  
Substantial Progress ◽  
Agonist And Antagonist ◽  
Antagonist Action ◽  
Polar Functional Groups ◽  
G Protein Coupled

AbstractG-protein coupled receptors are important pharmacological targets. Despite substantial progress, important questions still remain concerning the details of activation: how can a ligand act as an agonist in one receptor, but as an antagonist in a homologous receptor, and how can agonists activate a receptor despite lacking polar functional groups able to interact with helix 5? Studying vortioxetine, an important multimodal antidepressant drug, may elucidate both questions. Herein, we present a thorough in silico analysis of vortioxetine binding to 5-HT1A, 5-HT1B, and 5-HT7 receptors and compare to available experimental data. We are able to rationalize the differential mode of action of vortioxetine at different receptors, but also, in the case of the 5-HT1A receptor, we observe the initial steps of activation suggesting that interaction with helix 5 does not necessarily require a hydrogen bond as previously suggested. The results extend our current understanding of agonist and antagonist action at GPCRs.

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