Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. I. Coupling of alpha 2-adrenergic receptor subtypes to distinct G-proteins.
alpha 2-Adrenergic receptor (alpha 2-AR) subtypes couple to pertussis toxin (PT)-sensitive G-proteins to elicit both stimulatory and inhibitory cell responses. Signal specificity may be generated by the ability of the receptor subtypes to "recognize" distinct G-proteins with different affinity. To address this issue we stably expressed three alpha 2-AR subtypes, RNG alpha 2 (alpha 2B-AR), RG10 (alpha 2C-AR), and RG20 (alpha 2D-AR), in NIH-3T3 fibroblasts, which express two PT-sensitive G-proteins (Gi alpha 2, Gi alpha 3), and analyzed receptor/G-protein interactions by determining: 1) functional coupling to adenylylcyclase and 2) the ability of the receptors to exist in a high affinity state for agonist. In alpha 2D-AR transfectants expressing 200 or 2,200 fmol of receptor/mg of protein, epinephrine (10 microM) inhibited forskolin-induced elevation of cellular cAMP by 26 +/- 4.8% and 72 +/- 6.2%, respectively. Similar results were obtained in alpha 2B-AR transfectants. However, in alpha 2C-AR transfectants (200 fmol/mg) the forskolin-induced elevation of cellular cAMP was not altered by agonist treatment. In alpha 2C-AR transfectants expressing higher receptor densities (650-1,200 fmol/mg), epinephrine inhibited the effect of forskolin by 30 +/- 3.2%. This difference in functional coupling among the alpha 2-AR subtypes is reflected at the receptor/G-protein interface. In membrane preparations of alpha 2B and alpha 2D-AR but not alpha 2C-AR transfectants, agonist competition curves were biphasic, indicating high and low affinity states of the receptor for agonist. The high affinity state was guanyl-5'-yl imidodiphosphate- and PT-sensitive, indicative of receptor/G-protein coupling. These data suggest that the alpha 2C-AR differs from the alpha 2B and alpha 2D-AR subtypes in its ability to recognize PT-sensitive G-proteins expressed in NIH-3T3 fibroblasts. The alpha 2C-AR may couple preferentially to PT-sensitive G-proteins (Gi1, Go1,2) not expressed in NIH-3T3 fibroblasts and thereby elicit different cellular responses.
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Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
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Downing
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Lanier
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D016475:3T3 Cells; D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D000818:Animals; D000068438:Brimonidine Tartrate; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D015016:Yohimbine / Q000378:metabolism
D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D015016:Yohimbine; D006165:Guanylyl Imidodiphosphate; D000068438:Brimonidine Tartrate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D004837:Epinephrine