Amphipathic alpha-helical structure does not predict the ability of receptor-derived synthetic peptides to interact with guanine nucleotide-binding regulatory proteins.
In guanine nucleotide-binding regulatory protein- (G protein) coupled receptors, an amphipathic alpha-helix has been postulated to be the common structural determinant in the NH2- and COOH-terminal portions of the third intracellular loop representing the major interaction site with the G proteins. Here we assessed the ability of six peptides derived from these sites of the human dopamine D1-, D2-, and beta 1-adrenergic receptors to either activate G proteins directly or to uncouple the activity of their respective receptors in a native membrane environment. In addition, the cross-reactivity was analyzed. Nonspecific effects occurring at high concentrations were differentiated from G protein-specific effects. The peptide D2N derived from the NH2-terminal part of the third intracellular loop of the dopamine D2 receptor was the only one capable of specifically reversing the action of its receptor, the dopamine-mediated inhibition of the adenylyl cyclase. Furthermore, only D2N stimulated pertussis toxin-sensitive G proteins. However, D2N as the only peptide exhibiting specific effects did not exhibit the predicted amphipathic alpha-helix observed for mastoparan (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186) as demonstrated by circular dichroism spectroscopy. In contrast, a peptide for which a certain degree of helicality was verified spectroscopically (D1C) was neither active in GTPase and adenylyl cyclase determinations, nor did it block the receptor-mediated cyclase activation. Hence, the amphipathic alpha-helix does not represent the main structural determinant for the receptor-G protein interaction site.
Ernst Boehringer Institut für Arzneimittelforschung, Department of Protein Chemistry, Vienna, Austria.
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Voss
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Wallner
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Czernilofsky
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Freissmuth
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011487:Protein Conformation; D011943:Receptors, Adrenergic, beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017447:Receptors, Dopamine D1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D010446:Peptide Fragments; D011943:Receptors, Adrenergic, beta; D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases