Wild-type G Protein-coupled Receptors Research Articles

Molecular chaperones and G protein-coupled receptor maturation and pharmacology

G protein-coupled receptors (GPCRs) are highly conserved versatile signaling molecules located at the plasma membrane that respond to diverse extracellular signals. They regulate almost all physiological processes in the vertebrates. About 35% of current drugs target these receptors. Mutations in these genes have been identified as causes of numerous diseases. The seven transmembrane domain structure of GPCRs implies that the folding of these transmembrane proteins is extremely complicated and difficult. Indeed, many wild type GPCRs are not folded optimally. The most common defect in genetic diseases caused by GPCR mutations is misfolding and failure to reach the plasma membrane where it functions. General molecular chaperones aid the folding of all proteins, including GPCRs, by preventing aggregation, promoting folding and disaggregating small aggregates. Some GPCRs need additional receptor-specific chaperones to assist their folding. Many of these receptor-specific chaperones interact with additional receptors and alter receptor pharmacology, expanding the understanding of these chaperone proteins.

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization. However, the screening of fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel, selective, high affinity antagonists of human β2 adrenoceptor.

Two Ways to Be Sweet

The T1R family of G protein-coupled receptors (GPCRs), which are expressed in taste buds, detect sweet and umami tastes. Coexpressed T1R1 and T1R3 respond to the umami taste of glutamate, whereas coexpressed T1R2 and T1R3 respond to numerous sweet substances, including sugars, amino acids, proteins, and synthetic sweeteners. In humans, aspartame, neotame, and cyclamate stimulate T1R2-T1R3, whereas rat T1R2-T1R3 does not respond to these sweeteners. Xu et al. constructed rat-human chimeric T1R2 and T1R3 proteins in which the N-terminal region (containing the large extracellular "Venus flytrap" domain) from one species was fused to the C-terminal region (containing the transmembrane and intracellular domains) of the other. Analysis of various chimeric and human or rat wild-type GPCR combinations expressed in HEK-293 cells implicated the T1R2 N-terminal region in sensing aspartame and neotame, whereas extracellular portions of the C-terminal region of T1R3 were implicated in sensing cyclamate and in sensitivity to lactisole, a human-specific sweet taste inhibitor. Lactisole inhibited the response to glutamate (and glutamate plus the umami taste enhancer 5′-inosine monophosphate) both in vitro and in human taste tests, whereas cyclamate (whose sweetness would have overwhelmed an umami taste test) enhanced the T1R2-T1R3 response in vitro. Thus, the authors conclude that both T1R2 and T1R3 are required for a functional sweet taste receptor and that the structural diversity of sweeteners may depend in part on the existence of distinct ligand-binding sites. H. Xu, L. Staszewski, H. Tang, E. Adler, M. Zoller, X. Li, Different functional roles of T1R subunits in the heteromeric taste receptors. Proc. Natl. Acad. Sci. U.S.A. 101 , 14258-14263 (2004). [Abstract] [Full Text]

Five-transmembrane domains appear sufficient for a G protein-coupled receptor: functional five-transmembrane domain chemokine receptors.

The putative seven-transmembrane (TM) domains have been the structural hallmark for the superfamily of heterotrimeric G protein-coupled receptors (GPCRs) that regulate a variety of cellular functions by mediating a large number of extracellular signals. Five-TM GPCR mutants of chemokine receptor CCR5 and CXCR4, the N-terminal segment of which connected directly to TM3 as a result of a deletion of TM1-2 and the first intracellular and extracellular loops, have been obtained in this study. Laser confocal microscopy and flow cytometry analysis revealed that these five-TM mutant GPCRs were expressed stably on the cell surface after transfection into human embryonic kidney 293 cells. The five-TM CCR5 and CXCR4 functioned as normal chemokine receptors in mediating chemokine-stimulated chemotaxis, Ca2+ influx, and activation of pertussis toxin-sensitive G proteins. Like the wild-type GPCRs, the five-TM mutant receptors also underwent agonist-dependent internalization and desensitization and were subjected to regulation by GPCR kinases and arrestins. Our study indicates that five-TM domains, at least in the case of CCR5 and CXCR4, appear to meet the minimum structural requirements for a functional GPCR and suggests possible existence of functional five-TM GPCRs in nature during evolution.