Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization

• Published in: The Journal of biological chemistry 1993-09, Vol.268 (27), p.20285-20290
• Main Authors: BENYA, R. V, FATHI, Z, BATTEY, J. F, JENSEN, R. T
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding, Competitive; Biological and medical sciences; Bombesin - metabolism; Bombesin - pharmacology; Cell receptors; Cell structures and functions; CHO Cells; Cloning, Molecular; Cricetinae; DNA - genetics; DNA - metabolism; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Inositol 1,4,5-Trisphosphate - metabolism; Kinetics; Mice; Molecular and cellular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Receptors, Bombesin; Receptors, Neurotransmitter - biosynthesis; Receptors, Neurotransmitter - genetics; Receptors, Neurotransmitter - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Sequence Deletion; Serine; Threonine; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(20)80726-1

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.