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Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization
R V Benya1, Z Fathi, J F Battey
1Digestive Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Most seven-transmembrane G-protein-coupled receptors are rapidly internalized after binding agonist, but the general amino acid recognition sequences mediating this phenomenon have not been identified. In this study, components of the gastrin-releasing peptide receptor (GRP-R) regulating internalization were identified. Four GRP-R mutants with stop codons placed at variable distances distal to the putative palmitoylation sites Cys340-341 were transiently expressed in CHOP fibroblasts. A construct with a minimal carboxyl tail deletion, T375, bound and internalized agonist similarly to wild type receptor. Progressively larger truncations of the carboxyl terminus, however, increasingly impaired GRP-R-mediated internalization without altering receptor-agonist affinity. Three additional constructs were created: one with the putative palmitoylation sites replaced with Ala (CC340-341AA), one with the carboxyl-terminal protein kinase C-consensus sequence converted to Ala (TS360-361AA), and one with all Ser and Thr distal to Cys341 converted to Ala, Asn, or Gly (JF1). All constructs bound agonist similarly to wild type receptor. CC340-341AA internalized similarly to native receptor (93 +/- 3% of wild type by 60 min), whereas internalization of TS360-361AA was partially attenuated (64 +/- 2% of wild type by 60 min). JF1, however, internalized as poorly as T346, with only 16 +/- 2% of the wild type receptors internalized by 60 min. To assess G-protein coupling, selected receptor constructs were stably transfected into Balb fibroblasts, and phosphoinositol hydrolysis was determined. The largest GRP-R truncation, T346, increased total inositol phosphates (EC50 = 2.9 +/- 0.9 nM) similarly to wild type receptor (EC50 = 5.1 +/- 2.2 nM), as did CC340-341AA (EC50 = 5.4 +/- 1.5 nM) and TS360-361AA (EC50 = 3.1 +/- 1.2 nM). These data demonstrate that the multiple Ser and Thr located within the GRP-R carboxyl terminus distal to Cys341, including but not limited to those within the protein kinase C-consensus sequence, specifically regulate GRP-R internalization rates independent of receptor-G-protein coupling.
Insights
Researchers identified key amino acid sequences in the gastrin-releasing peptide receptor (GRP-R) that control its internalization. Specific serine and threonine residues in the carboxyl terminus regulate GRP-R internalization independently of G-protein coupling.
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Biochemistry
Background:
- Seven-transmembrane G-protein-coupled receptors (GPCRs) are crucial cell surface proteins involved in numerous physiological processes.
- GPCR internalization following agonist binding is a key mechanism for signal termination and receptor regulation.
- The specific molecular determinants governing GPCR internalization, particularly within the receptor's carboxyl terminus, remain incompletely understood.
Purpose of the Study:
- To identify the specific amino acid sequences within the gastrin-releasing peptide receptor (GRP-R) carboxyl terminus that regulate its agonist-induced internalization.
- To determine whether these regulatory sequences affect GRP-R affinity for its agonist or its G-protein coupling.
- To elucidate the role of palmitoylation sites and protein kinase C (PKC) consensus sequences in GRP-R internalization.
Main Methods:
- Site-directed mutagenesis was used to create various GRP-R carboxyl-terminal mutants, including truncations and substitutions of key residues (e.g., palmitoylation sites, PKC sites).
- Mutant receptors were expressed in CHOP fibroblasts to assess agonist binding, internalization rates via radioligand binding assays, and G-protein coupling through phosphoinositol hydrolysis assays in stably transfected Balb fibroblasts.
- Quantitative analysis of receptor internalization and G-protein signaling was performed to correlate structural modifications with functional outcomes.
Main Results:
- Progressive truncation of the GRP-R carboxyl terminus significantly impaired agonist-induced internalization without affecting agonist binding affinity.
- Mutations at the palmitoylation sites (CC340-341AA) did not alter internalization, while modifications to the carboxyl-terminal Ser/Thr residues (JF1 mutant) severely attenuated internalization.
- Mutant receptors, including those with impaired internalization, exhibited normal G-protein coupling and phosphoinositol hydrolysis, indicating internalization regulation is independent of G-protein signaling.
- The JF1 mutant, lacking specific Ser/Thr residues distal to Cys341, showed internalization levels comparable to the severely truncated T346 mutant.
Conclusions:
- Multiple serine and threonine residues in the GRP-R carboxyl terminus, distal to Cys341, are critical for regulating receptor internalization rates.
- These regulatory sequences function independently of the receptor's ability to bind agonists or couple to G-proteins.
- The findings provide novel insights into the molecular mechanisms governing GPCR internalization and receptor trafficking.