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J. Biol. Chem., Vol. 280, Issue 25, 23464-23474, June 24, 2005

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Different Structural Requirements for the Constitutive and the Agonist-induced Activities of the ₂-Adrenergic Receptor^*

Caterina Ambrosio, Paola Molinari, Francesca Fanelli, Yoshiro Chuman¶||, Maria Sbraccia, Ozlem Ugur**, and Tommaso Costa

From the Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy, the Dulbecco Telethon Institute and Department of Chemistry, University of Modena, Modena 41100, Italy, the ^¶Laboratory of Structure-Function Biochemistry, Department of Chemistry, Kyusho University, Fukuoka, Japan, and the ^**Department of Pharmacology, Ankara University, Ankara 06339, Turkey

We converted Ser-207, located in helix 5 of the ₂-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to G_s activation, we expressed the mutants in a G_s-tethered form. GTP exchange in such constructs is restricted to the fused -subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.

Received for publication, March 16, 2005

^* This work was supported in part by the Ministero dell'Istruzione dell'Università e della Ricerca (Italy) Grant Fondo Italiano per la Ricerca di Base RBA01XWSA_003 and by the Mombushu Foundation (Japan) for a visiting fellowship (to Y. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Dept. of Biological Chemistry, Hokkaido University, Sapporo 060-0810, Japan.

To whom correspondence should be addressed. Tel.: 39-064-990-2386; Fax: 39-064-938-7104; E-mail: tomcosta{at}iss.it.

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