Fusion of beta 2-adrenergic receptor to G alpha s in mammalian cells: identification of a specific signal transduction species not characteristic of constitutive activation or precoupling.

The forward and antegrade interactions that comprise the agonist receptor-G protein complex were studied in Chinese hamster fibroblasts transfected to express the beta(2)-adrenergic receptor (beta(2)AR), the beta(2)AR and the alpha-subunit of its cognate G protein (G(s)), and a protein consisting of the beta(2)AR fused at its carboxy terminus with G(alpha)(s) (beta(2)AR-G(s)). Expression levels were matched at approximately 600 fmol/mg. Basal adenylyl cyclase activities were increased with the fusion receptor membranes compared to coexpressed receptor plus G(alpha)(s), and to wild-type beta(2)AR (20.5 +/- 1.8 vs 9.0 +/- 0.88 vs 8.7 +/- 0.93 pmol min(-)(1) mg(-)(1)), confirming in mammalian cells that the fusion of beta(2)AR and G(alpha)(s) results in a state not attained by expression of unfused components. However, agonist-stimulated activities were not increased proportionally, such that the stimulation over basal of the beta(2)AR-G(s) fusion protein (1. 5-fold) was less than wild-type beta(2)AR (2.1-fold). Agonist competition studies performed in the absence of guanine nucleotide exhibited high-affinity binding sites with a lower K(H) (1.75 vs 8. 47 nM) and greater %R(H) (51% vs 44%) for beta(2)AR-G(s), but GppNHp failed to convert most of these to the low-affinity state. Functional studies with the inverse agonist ICI 118551 did not show enhanced efficacy or potency with the fusion protein. Adenylyl cyclase studies with three partial agonists with diverse structures (dobutamine, ritodrine, and phenylephrine) showed no enhancement of efficacy with beta(2)AR-G(s) and a minor trend toward enhanced potency. Taken together, these results indicate that the tethering of G(alpha)(s) to the beta(2)AR causes a conformational change in the receptor that stabilizes a species "trapped" between the non-guanine nucleotide-bound state and the GTP-bound form. Functionally the receptor is not characterized by a consistent pattern of properties ascribed to other states such as constitutive activation or precoupling, but rather represents a unique state in the transition from high- to low-affinity forms.