Dominance of Gs in doubly Gs/Gi-coupled chimaeric A1/A2A adenosine receptors in HEK-293 cells

Abstract

A(1) adenosine receptors inhibit adenylate cyclase by activating G(i)/G(o), whereas A(2A) receptors activate G(s). We examined how regions of A(1) and A(2A) receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to G(i)/G(o) and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A(1)/A(2A) 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A(1) 3ICL sequence (A(2A)/A(1)L or A(2A)/A(1)LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to G(i)/G(o). These chimaeras have basal cAMP levels lower than those of wild-type A(2A) receptors, similar to wild-type A(1) receptors. The A(1) C-terminus modulates the coupling of receptors with A(1) 3ICL such that A(2A)/A(1)LT is better coupled to G(i)/G(o) than A(2A)/A(1)L. The C-terminus has little impact on coupling to receptors containing A(2A) 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to G(i)/G(o) mediated by A(1) 3ICL and not by other intracellular domains that favour G(i) coupling. The C-terminus sequence has little or no effect on coupling to G(s). For doubly G(s)/G(i)-coupled adenosine receptors in HEK-293 cells, G(s)-mediated stimulation predominates over G(i)/G(o)-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.

Expression of A3Adenosine Receptors in Human Lymphocytes: Up-Regulation in T Cell Activation

“…Most probably, its stimulatory effect on cAMP levels, raised through the activation of A 2A receptors, prevailed over the A 3 -mediated inhibitory action, suggesting a stronger potency of A 2A receptors in signal transduction over the A 3 receptors. This is not surprising, because a similar behavior has already been reported in doubly Gs/Gicoupled chimeric A 1 /A 2A adenosine receptors expressed in human embryonic kidney 293 cells (Tucker et al, 2000). Therefore, future studies will help to reconcile the complicated effects of adenosine through G i protein-coupled A 3 adenosine receptors in the modulation of immune and inflammatory processes and to clarify the relevance of the differential activation by adenosine of Gs/Gi coupled receptor subtypes to reach a balance between destruction of pathogen agents and tissue protection from excessive damage (Sitkovsky, 2003).…”

Steroid and G Protein Binding Characteristics of the Seatrout and Human Progestin Membrane Receptor α Subtypes and Their Evolutionary Origins

“…The activation of an inhibitory G protein by the mPR␣s is also consistent with our recent findings on the signaling pathways activated by 20␤-S during meiotic maturation of seatrout oocytes (25) as well as those activated by progesterone through the wild-type hu-mPR␣ in human myometrial cells (26). A unique characteristic of GPCRs is that treatment with nonhydrolyzable forms of guanine nucleotides such as GTP␥-S, and for G i/o -protein coupled receptors with pertussis toxin, cause uncoupling of G proteins from the receptors (45,46), resulting in a decrease in their ligand binding affinities (47,48). These treatments caused similar decreases in ligand binding to st-mPR␣ and humPR␣.…”

Animal models for the study of adenosine receptor function

“…Residues required for coupling to G-proteins may also vary between receptors. Whereas the 3rd intracellular loop and the C-terminus contribute to A1 receptor coupling to G-proteins, only the 3rd intracellular loop has been implicated in coupling of A2a to G-proteins (Tucker et al, 2000). Phosphorylation of intracellular loops also appears to be involved in desensitization of adenosine receptors (Palmer and Stiles, 1997a).…”