Abstract
Despite extensive study of heptahelical G protein-coupled receptors (GPCRs), the precise mechanism of G protein activation is unknown. The role of one highly conserved stretch of residues, the amino acids glutamic acid/aspartic acid–arginine–tyrosine (i.e., the E/DRY motif), has received considerable attention with respect to regulating GPCR conformational states. In the consensus view, glutamic acid/aspartic acid maintains the receptor in its ground state, because mutations frequently induce constitutive activity (CA). This hypothesis has been confirmed by the rhodopsin ground-state crystal structure and by computational modeling approaches. However, some class A GPCRs are resistant to CA, suggesting alternative roles for the glutamic acid/aspartic acid residue and the E/DRY motif. Here, we propose two different subgroups of receptors within class A GPCRs that make different use of the E/DRY motif, independent of the G protein type (Gs, Gi, or Gq) to which the receptor couples. In phenotype 1 receptors, nonconservative mutations of the glutamic acid/aspartic acid–arginine residues, besides inducing CA, increase affinity for agonist binding, retain G protein coupling, and retain an agonist-induced response. In contrast, in second phenotype receptors, the E/DRY motif is more directly involved in governing receptor conformation and G protein coupling/recognition. Hence, mutations of the glutamic acid/aspartic acid residues do not induce CA. Conversely, nonconservative mutations of the arginine of the E/DRY motif always impair agonist-induced receptor responses and, generally, reduce agonist binding affinity. Thus, it is essential to look beyond the rhodopsin ground-state model of conformational activation to clarify the role of this highly conserved triplet in GPCR activation and function.
Footnotes
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This work was supported in part by grants from the Italian Ministry of University and Research, MIUR–PRIN 2004 (prot. 2004052155) to G. E. Rovati and V. Capra.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.029470.
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ABBREVIATIONS: GPCR, heptahelical G protein-coupled receptors; CAM, constitutively active mutant; CA, constitutive activity; CIM, constitutively inactive mutant; CCR5, chemokine 5; GnRH, gonadotropin-releasing hormone; CB2R, cannabinoid 2; OT-R, oxytocin receptor; μO-R, μ-opioid receptors; H2R, histamine H2 receptors; V2R, vasopressin type II receptors; (α1B-AR, α1B-adrenergic receptor; TM, transmembrane domain; P1-type, first phenotype; P2-type, second phenotype; TP-R, TP receptor; V1AR, V1A receptor; AChR, acetylcholine receptor; GTPγS, guanosine 5′-O-(3-thio)triphosphate.
- Received August 24, 2006.
- Accepted December 20, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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