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Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function.

ABSTRACT: 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).

SUBMITTER: Fortin JP

PROVIDER: S-EPMC2683074 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function.

Fortin Jean-Philippe JP Zhu Yuantee Y Choi Charles C Beinborn Martin M Nitabach Michael N MN Kopin Alan S AS

Proceedings of the National Academy of Sciences of the United States of America 20090423 19

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 m ...[more]

PMID: 19416829

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Project description:The ligands of certain G-protein coupled receptors (GPCRs) are membrane soluble and reach their target from the lipid bilayer. Lipid composition and dynamics will therefore modulate the activity of these receptors, but specific roles of lipid components, including the ubiquitous cholesterol (Chol), are not clear. We have probed the organization and dynamics of such a lipid-bilayer-penetrating ligand, the endogenous ligand for the κ-opioid receptor (KOR) dynorphin A (1-17) (DynA), using molecular dynamics (MD) simulations of DynA in cholesterol-depleted and cholesterol-enriched model membranes. DynA is found to penetrate deep inside fluid dimyristoylphosphatidylcholine (DMPC) bilayers, and resides with its N-terminal helix at ∼6 Å away from the bilayer midplane, in a tilted orientation, at an ∼50° angle with respect to the membrane normal. In contrast, DynA inside DMPC/Chol membranes with 20% cholesterol (DMPC/Chol) is situated with its helical segment ∼5 A higher, i.e., closer to the lipid/water interface and in a relatively vertical orientation. The DMPC membrane shows greater thinning around the insertion and permits a stronger influx of water inside the hydrocarbon core than the DMPC/Chol membranes. Relating these results to data about key GPCR residues that have been implicated in interactions with membrane-inserting GPCR ligands, we conclude that the position of DynA in DMPC/Chol, but not in pure DMPC, correlates with generally proposed GPCR ligand entry pathways. Our predictions provide a possible mechanistic explanation as to why DynA binding to KOR, and the subsequent activation of the receptor, is facilitated in cholesterol-enriched environments. A quantitative description of DynA-induced membrane deformations is obtained with a continuum theory of membrane deformations (CTMD) that is based on hydrophobic matching. Comparison with the MD data reveals the significance of the lipid tail packing energy contribution in the DMPC/Chol mixtures in predicting equilibrium membrane shape around DynA. On this basis, specific corrections are introduced to this energy term within the CTMD framework, thereby extending the applicability of the CTMD framework to lipid raft mixtures and their interactions with GPCR proteins and their ligands.

Dataset Information

Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function.

Publications

Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function.

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