SERINES AND THREONINES IN THE GASTRIN-RELEASING PEPTIDE RECEPTOR CARBOXYL-TERMINUS MEDIATE INTERNALIZATION

Most seven-transmembrane G-protein-coupled receptors are rapidly inter nalized 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) re gulating internalization were identified. Four GRP-R mutants with stop codons placed at variable distances distal to the putative palmitoyla tion sites Cys340-341 were transiently expressed in CHOP fibroblasts. A construct with a minimal carboxyl tail deletion, T375, bound and int ernalized agonist similarly to wild type receptor. Progressively large r truncations of the carboxyl terminus, however, increasingly impaired GRP-R-mediated internalization without altering receptor-agonist affi nity. Three additional constructs were created: one with the putative palmitoylation sites replaced with Ala (CC340-341 AA), one with the ca rboxyl-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 w ild type receptor. CC340-341AA internalized similarly to native recept or (93 +/- 3% of wild type by 60 min), whereas internalization of TS36 0-361AA was partially attenuated (64 +/- 2% of wild type by 60 min). J F1, however, internalized as poorly as T346, with only 16 +/- 2% of th e wild type receptors internalized by 60 min. To assess G-protein coup ling, selected receptor constructs were stably transfected into Balb f ibroblasts, 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 l ocated within the GRP-R carboxyl terminus distal to Cys341, including but not limited to those within the protein kinase C-concensus sequenc e, specifically regulate GRP-R internalization rates independent of re ceptor-G-protein coupling.