Dominance of G(s) in doubly G(s)/G(i)-coupled chimaeric A(1)/A(2A) adenosine receptors in HEK-293 cells.
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.
Tucker
A L
AL
Department of Internal Medicine (Cardiovascular Division), Box 6012, MR4 Building, University of Virginia Health System, Charlottesville, VA 22908, USA. alt8t@virginia.edu
Jia
L G
LG
Holeton
D
D
Taylor
A J
AJ
Linden
J
J
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D037361:Adenylate Cyclase Toxin; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin; D010627:Phenethylamines / Q000494:pharmacology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D018076:DNA, Complementary; D008024:Ligands; D010627:Phenethylamines; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D005576:Colforsin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000241:Adenosine