Abstract
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.
Highlights
calcium-sensing receptor (CaR) is a member of the family 3 G-protein-coupled receptors (GPCRs) gene family that includes eight metabotropic glutamate receptors, two ␥-aminobutyric acid receptor subunits (GABAB1 and GABAB2), three sweet and umami taste receptors (T1R1, T1R2, and T1R3), several putative rodent pheromone receptors (V2Rs), and orphan receptors (GPRC6A, GPRC5B–5D) [2]
3 G-protein-coupled receptors (GPCRs), which includes metabotropic glutamate receptors, 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
We examined the peptide linker region connecting the ECD to the 7TMD of a prototypical member of the family 3 GPCR, the CaR, to explore whether this sequence plays a role in the activation of the 7TMD upon binding of ligand in the extracellular Venus flytrap domain motif (VFTM) of this receptor
Summary
GPCRs [9, 10]. To address how the ligand-induced conformational changes of the VFTM might be transmitted for G-protein coupling, the peptide linkers of the GABAB receptor heterodimer were examined [11]. Modification of two GABAB receptor subunit linkers by changes in sequence and/or length were mostly tolerated and the linker regions in GABAB receptors were predicted to act only as tethers for the VFTMs to the 7TMD, supporting a direct contact model of receptor activation. In this model illustrated, a, receptor activation occurs predominantly through contacts between the ligandbound VFTM and exo-loops of the 7TMD and the linker acts solely to keep the VFTM in proximity to the 7TMD. Our analysis of the expression and signaling properties of these receptor constructs suggests an essential role for highly conserved amino acids within this structure
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