Yoshiro Chuman

Different Structural Requirements for the Constitutive and the Agonist-induced Activities of the β2-Adrenergic Receptor

We converted Ser-207, located in helix 5 of the β2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to Gs activation, we expressed the mutants in a Gαs-tethered form. GTP exchange in such constructs is restricted to the fused α-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.