Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function
| Autor: | Yuantee Zhu, Martin Beinborn, Michael N. Nitabach, Alan S. Kopin, Charles Choi, Jean-Philippe Fortin |
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| Rok vydání: | 2009 |
| Předmět: |
Peptide Hormones
C-C chemokine receptor type 7 Secretin family Biology Ligands Biochemistry Incretins Models Biological Cell Line Receptors G-Protein-Coupled Enzyme-linked receptor Humans Point Mutation Glucose homeostasis 5-HT5A receptor Receptor G protein-coupled receptor Multidisciplinary Cell Membrane Biological Sciences Protein Structure Tertiary Cell biology Transmembrane domain Peptides Gene Deletion |
| Zdroj: | Proceedings of the National Academy of Sciences. 106:8049-8054 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Class B1 (secretin family) G protein-coupled receptors (GPCRs) modulate a wide range of physiological functions, including glucose homeostasis, feeding behavior, fat deposition, bone remodeling, and vascular contractility. Endogenous peptide ligands for these GPCRs are of intermediate length (27–44 aa) and include receptor affinity (C-terminal) as well as receptor activation (N-terminal) domains. We have developed a technology in which a peptide ligand tethered to the cell membrane selectively modulates corresponding class B1 GPCR-mediated signaling. The engineered cDNA constructs encode a single protein composed of ( i ) a transmembrane domain (TMD) with an intracellular C terminus, ( ii ) a poly(asparagine-glycine) linker extending from the TMD into the extracellular space, and ( iii ) a class B1 receptor ligand positioned at the N terminus. We demonstrate that membrane-tethered peptides, like corresponding soluble ligands, trigger dose-dependent receptor activation. The broad applicability of this approach is illustrated by experiments using tethered versions of 7 mammalian endogenous class B1 GPCR agonists. In parallel, we carried out mutational studies focused primarily on incretin ligands of the glucagon-like peptide-1 receptor. These experiments suggest that tethered ligand activity is conferred in large part by the N-terminal domain of the peptide hormone. Follow-up studies revealed that interconversion of tethered agonists and antagonists can be achieved with the introduction of selected point mutations. Such complementary receptor modulators provide important new tools for probing receptor structure–function relationships as well as for future studies aimed at dissecting the tissue-specific biological role of a GPCR in vivo (e.g., in the brain vs. in the periphery). |
| Databáze: | OpenAIRE |
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