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.
Highlights
Most seven-transmembrane G-protein-coupled re- protein1-coupled receptors becomes modulated, a phenomeceptors are rapidly internalized after binding agonist, non referred to as desensitization
Madison,WI).Thestructure of themutatedGRP-RcDNA was sponsecurves for bombesin binding to that seen with the confirmed by dideoxy sequencing [33]
To determine whether the Gastrin-releasing peptide (GRP)-R carboxyl terminus was involved in influencing internalization rates,we determined the abilityof the four truncated mutants and the philicity profile for the cytoplasmic tail of JF1 as for the wild type wild type receptor to internali[z'2e51-Tyr4]bombesinin CHOP
Summary
Most seven-transmembrane G-protein-coupled re- protein1-coupled receptors becomes modulated, a phenomeceptors are rapidly internalized after binding agonist, non referred to as desensitization. ~ 3 4,8with only 16 f 2% of the wild type receptors zation rates [6],mutants whose cytoplasmic tail has been internalized by 60 min.
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