Elucidation of the role of peptide linker in calcium-sensing receptor activation process.
Family 3 G-protein-coupled receptors (GPCRs), which includes metabotropic glutamate receptors (mGluRs), sweet and "umami" taste receptors (T1Rs), and the extracellular calcium-sensing receptor (CaR), represent a distinct group among the superfamily of GPCRs characterized by large amino-terminal extracellular ligand-binding domains (ECD) with homology to bacterial periplasmic amino acid-binding proteins that are responsible for signal detection and receptor activation through as yet unresolved mechanism(s) via the seven-transmembrane helical domain (7TMD) common to all GPCRs. To address the mechanism(s) by which ligand-induced conformational changes are conveyed from the ECD to the 7TMD for G-protein activation, we altered the length and composition of a 14-amino acid linker segment common to all family 3 GPCRs except GABA(B) receptor, in the CaR by insertion, deletion, and site-directed mutagenesis of specific highly conserved residues. Small alterations in the length and composition of the linker impaired cell surface expression and abrogated signaling of the chimeric receptors. The exchange of nine amino acids within the linker of CaR with the homologous sequence of mGluR1, however, preserved receptor function. Ala substitution for the four highly conserved residues within this amino acid sequence identified a Leu at position 606 of the CaR critical for cell surface expression and signaling. Substitution of Leu(606) for Ala resulted in impaired cell surface expression. However, Ile and Val substitutions displayed strong activating phenotypes. Disruption of the linker by insertion of nine amino acids of a random-coiled structure uncoupled the ECD from regulating the 7TMD. These data are consistent with a model of receptor activation in which the peptide linker, and particularly Leu(606), provides a critical interaction for the CaR signal transmission, a finding likely to be relevant for all family 3 GPCRs containing this conserved motif.
Ray
Kausik
K
Laboratory of Cellular Biology, NIDCD, National Institutes of Health, Bethesda, Maryland 20892,USA. rayk@nidcd.nih.gov
Adipietro
Kaylin A
KA
Chen
Claudia
C
Northup
John K
JK
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D020013:Calcium Signaling / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism*
D044169:Receptors, Calcium-Sensing