Identification of A(2a) adenosine receptor domains involved in selective coupling to Gs: Analysis of chimeric A1/A(2a) adenosine receptors

Abstract

Responses to adenosine are governed by selective activation of distinct G proteins by adenosine receptor (AR) subtypes. The A(2a)AR couples via G(s) to adenylyl cyclase stimulation while the A1AR couples to G1 to inhibit adenylyl cyclase. To determine regions of the A(2a)AR that selectively couple to G(s), chimeric A1/A(2a)ARs were expressed in Chinese hamster ovary cells and ligand binding and adenylyl cyclase activity analyzed. Replacement of the third intracellular loop of the A(2a)AR with that of the A1AR reduced maximal adenylyl cyclase stimulation and decreased agonist potency. Restricted chimeras indicated that the NH2-terminal portion of intracellular loop 3 was predominantly responsible for this impairment. Reciprocal chimeras composed primarily of A1AR sequence with limited A(2a)AR sequence substitution stimulated adenylyl cyclase and thus supported these findings. A lysine and glutamic acid residue were identified as necessary for efficient A(2a)AR-G(s) coupling. Analysis of chimeric receptors in which sequence of intracellular loop 2 was substituted indicated that the nature of amino acids in this domain may indirectly modulate A(2a)AR-G(s) coupling. Replacement of the cytoplasmic tall of the A(2a)AR with the A1AR tail did not affect adenylyl cyclase stimulation. Thus, selective activation of G(s) is predominantly dictated by the NH2-terminal segment of the third intracellular loop of the A(2a)AR.