To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Mutational and computational analysis of the alpha-1b adrenergic receptor.Involvement of basic and hydrophobic residues in receptor activation and G protein coupling / P. J., Greasley; Fanelli, Francesca; A., Scheer; L., Abuin; M., NENNINGER TOSATO; DE BENEDETTI, Pier Giuseppe; S., Cotecchia. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - ELETTRONICO. - 276:49(2001), pp. 46485-46494. [10.1074/jbc.M105791200]

Mutational and computational analysis of the alpha-1b adrenergic receptor.Involvement of basic and hydrophobic residues in receptor activation and G protein coupling

FANELLI, Francesca;DE BENEDETTI, Pier Giuseppe;
2001

Abstract

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.
2001
276
49
46485
46494
Mutational and computational analysis of the alpha-1b adrenergic receptor.Involvement of basic and hydrophobic residues in receptor activation and G protein coupling / P. J., Greasley; Fanelli, Francesca; A., Scheer; L., Abuin; M., NENNINGER TOSATO; DE BENEDETTI, Pier Giuseppe; S., Cotecchia. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - ELETTRONICO. - 276:49(2001), pp. 46485-46494. [10.1074/jbc.M105791200]
P. J., Greasley; Fanelli, Francesca; A., Scheer; L., Abuin; M., NENNINGER TOSATO; DE BENEDETTI, Pier Giuseppe; S., Cotecchia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/307851
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