BETA-ADRENERGIC-RECEPTOR ASSAY IN PERMEABLE CELLS

We developed a method for assaying agonist binding to study the coupling of beta-adrenergic receptors to G proteins in an environment that resembles an intact cell. This method, devised using sapinin-permeable cell suspensions, permits investigation of beta-adrenergic receptor behavior under conditions where the receptor appears to be tightly coupled to Os protein to activate adenylyl cyclase. In this communication, we compare the binding characteristics of beta-adrenergic receptors in permeable cell suspensions and cell membrane preparations. The cell lines examined here, by means of [I-125]pindolol binding, were C6 glioma cells and NCB hybrid cells. Scatchard plots for permeable cells were linear with correlation coefficients greater than 0.97 and the best fit was by the single site model. Furthermore, Bmax values of C6 cells and NCB cells were consistent with those of membranes. However, IC50 values of isoproterenol competition for [I-125]pindolol binding sites and the extent of GppNHp-induced changes of IC50 were not identical in permeable cells and cell membranes, suggesting that beta-adrenergic receptors behave differentially in the undisrupted cellular milieu than in a membrane preparation. Our newly devised method permits one to obtain further information about receptor coupling in a nearly intact cell.