Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization.
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.
Digestive Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
R V
RV
Benya
Z
Z
Fathi
J F
JF
Battey
R T
RT
Jensen
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001839:Bombesin / Q000378:metabolism* / Q000494:pharmacology*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D012694:Serine; D013912:Threonine; D014162:Transfection
D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA; D001839:Bombesin