G(i) activator region of alpha(2A)-adrenergic receptors: distinct basic residues mediate G(i) versus G(s) activation.
The structural determinants of G protein coupling versus activation by G protein-coupled receptors are not well understood. We examine the role of two distinct basic regions in the carboxyl terminal portion of the third intracellular loop of the alpha(2A)-adrenergic receptor to dissect these aspects of function. Changing three arginines to alanines by mutagenesis and stable expression in Chinese hamster ovary-K1 cells impaired the alpha(2)-adrenergic receptor G(s)-mediated stimulation of cyclic AMP (cAMP) accumulation, whereas G(i)-mediated inhibition was normal. When two (B2) or three (B3) basic residues closer to transmembrane span 6 were mutated to alanine, normal ligand binding was observed, but G(i)-mediated inhibition of cAMP accumulation showed 20-fold and 50-fold decreases in agonist potency for the B2 and B3 mutants, respectively. Surprisingly, a normal G(s) response was seen for the B2 mutant, and the B3 mutant showed only a 6-fold decrease in agonist potency. Mutation of both the three alanines and B3 residues to alanines showed a 200-fold decrease in agonist potency for G(i)-mediated inhibition of cAMP accumulation, whereas the G(s) response was nearly completely eliminated. The three basic residues (which include the BB of the BBXXF motif) play a role as G(i) activators rather than in receptor-G protein coupling, because high-affinity agonist binding is intact. Thus, we have identified three basic residues required for activation of G(i) but not required for receptor-G protein coupling. Also, distinct basic residues are required for optimal G(i) and G(s) responses, defining a microspecificity determinant within the carboxyl terminal portion of the third intracellular loop of the alpha(2a) adrenergic receptor.
Wade
S M
SM
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan, USA.
Lim
W K
WK
Lan
K L
KL
Chung
D A
DA
Nanamori
M
M
Neubig
R R
RR
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*
D008565:Membrane Proteins; D018341:Receptors, Adrenergic, alpha-2; D001120:Arginine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs