Year-end Sale % OFF 
Abstract
Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized alphaT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser153 with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser151, Ser153, and Thr142) or only Ser153 was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser153) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in alphaT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser153 operates through a phosphorylation-independent mechanism.
Highlights
The desensitization mechanism of GPCRs1 involves binding of the agonist to the receptor, which causes activation of the effector and facilitates phosphorylation of the receptor by second messengers-activated kinases or by G-proteincoupled receptor kinases (GRKs) on specific sites within the C-terminal tail
Synthetic Peptides Corresponding to ICL2 and ICL3 but Not ICL1 of the GnRHR Potentiate inositol phosphate (InsP) Formation by gonadotropin-releasing hormone (GnRH)—As mentioned before, one of the unique structural features of the mammalian type I GnRHR is the absence of a C-terminal tail
To test the hypothesis that the intracellular loops (ICLs) may be involved in GnRHR regulation, synthetic peptides corresponding to the ICLs of the GnRHR were synthesized
Summary
Materials—The stable GnRH agonist Buserelin was used throughout this study and was kindly provided by Dr J. Cells were washed twice with PBS and incubated with 2 ml/well PBS containing 20 g/ml plasmid DNA and 0.2 mg/ml DEAE-dextran for 30 min at 37 °C. The cells were washed three times with DMEM containing 0.1% bovine serum albumin and incubated for 15 min at 37 °C with 1 ml of the same buffer containing 10 mM LiCl. The cells were treated with the corresponding concentration of GnRH. Binding Assay—Cells (5 ϫ 106/well) were washed three times with the assay buffer (PBS containing 0.1% bovine serum albumin (pH 7.4)) and incubated for 60 min at room temperature with 125I-labeled GnRH-A (100,000 cpm/ml) and increasing concentrations of unlabeled GnRH-A. The pellet was collected and resuspended in sample buffer for protein separation on 10% SDS-PAGE, followed by Western blotting with mouse monoclonal antibody directed at the HA epitope tag [10]. The blots were autoradiographed on Kodak X-100 films, and the phosphorylation was quantitated by densitometry (Bio-Rad 690 densitometer)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
