A M Koachman

We have used wild type 549 lymphoma cells and S49 variants that have lesions in their ability to generate CAMP to explore the mechanism of activation of adenylate cyclase by the diterpene forskolin.In intact wild type cells, forskolin rapidly and reversibly stimulated CAMP accumulation several hundred-fold over basal levels.Simultaneous addition of forskolin and the padrenergic agonist isoproterenol gave greater than additive (Le.synergistic) responses.Forskolin lowered the Kact for isoproterenol-stimulated CAMP accumulation 2-to %fold and increased maximal response to isoproterenol 8-to 10-fold; isoproterenol decreased the K,,, for forskolin about 10-fold but had a much smaller effect (t2-fold) on maximal response.In competitive binding studies between (-)isoproterenol and [1261]iodocyanopindolol, forskolin more than doubled the fi-action of /%adrenergic receptors in a high affinity (-5-10

We have used wild-type and variants of the T-lymphoma cell line S49 to explore internalization and down-regulation of adenylate cyclase-linked beta-adrenergic receptors. Internalization was defined by the loss in "surface receptors" detected at 4 degrees C on intact cells by the antagonists [3H]CGP-12177 or [125I]iodocyanopindolol, whereas down-regulation was defined as the loss in total cellular content of receptors [( 125I]iodocyanopindolol binding assayed at 37 degrees C). In wild-type cells, the beta-adrenergic agonist isoproterenol induced a rapid (t 1/2, approximately equal to 1 min) and reversible loss in surface receptors. The surface sites were lost at a rate similar to the rate of desensitization of beta-adrenergic receptor-mediated cyclic AMP generation of S49 cells. A series of S49 variants (cyc-, UNC, H21a) having lesions in NS (the guanine nucleotide binding protein that couples beta-receptors to adenylate cyclase) or with absent cAMP-dependent protein kinase activity (kin-), had a loss in surface sites that was equivalent to that of wild-type cells. By contrast, S49 variant cells having lesions in NS showed variable rates and extents of down-regulation of beta-adrenergic receptors. In wild-type and kin- S49 cells, beta-receptors down-regulated with a t 1/2 of approximately equal to 4 hr. Down-regulation was blunted in the cyc- and UNC variants that have altered coupling of receptors to NS, but it was faster in the H21a variant that retains receptor-NS interaction. Recovery of receptors after down-regulation occurred at a similar rate (t 1/2, approximately equal to 6 hr) in wild-type, UNC, and H21a cells. These results demonstrate that internalization of beta-adrenergic receptors may be necessary, but is not sufficient, to explain agonist-induced receptor down-regulation in S49 cells. The variable expression in the development of down-regulation in S49 variants implies that receptor-NS interaction regulates the fate of receptors linked to the stimulation of adenylate cyclase.

Studies of beta-adrenergic receptors on several types of intact target cells indicate that agonists, but not antagonists, show prominent KD/Kact discrepancies--lower affinities (KD) in equilibrium binding studies (i.e. in competition with antagonist radioligands) than in functional assays (Kact). In this report we show that intact S49 lymphoma cells initially bind beta-adrenergic agonists in a high affinity manner but that this high affinity binding rapidly converts to a low affinity state. This time course is similar to that of agonist-mediated desensitization in S49 cells. In competitive binding studies conducted with [125I]iodocyanopindolol or [125I]iodohydroxybenzylpindolol, IC50 values for beta-adrenergic agonists increased 13-200-fold between 1 and 60 min, whereas antagonists yielded similar IC50 values at the two time points. The binding of antagonists, but not agonists, could be simulated by a computer model based on the law of mass action. In contrast with results in intact S49 cells, crude membrane fractions yielded similar IC50 values for agonists in 1- and 60-min incubations with [125I]iodocyanopindolol. Moreover, time-dependent decreases in apparent affinity of the agonist (-)-isoproterenol were observed not only in wild type S49 cells but also in several S49 variants (UNC, cyc-, H21a) having defects in Ns, the guanine nucleotide binding protein that couples receptors to activation of adenylate cyclase, and in Kin-, an S49 variant with absent cAMP-dependent protein kinase activity. These results show that beta-adrenergic receptors of intact S49 cells demonstrate a prominent time-dependent decrease in apparent affinity for agonists and that this decrease in affinity does not require cAMP generation, the Ns components defective in several S49 variants or cAMP-dependent protein kinase. The rapid conversion of agonist binding from high to low affinity can account for the rapid desensitization of intact cells to catecholamines and probably explains previously reported KD/Kact discrepancies of intact cells.

Studies of @-adrenergic receptors on several types of intact target cells indicate that agonists, but not antagonists, show prominent KDIK,,~ discrepancies-lower affinities (KD) in equilibrium binding studies (Le. in competition with antagonist radioligands) than in functional assays (Kact). In this report we show that intact S49 lymphoma cells initially bind @-adrenergic agonists in a high affinity manner but that this high affinity binding rapidly converts to a low affinity state. This time course is similar to that of agonist-mediated desensitization in 549 cells. In competitive binding studies conducted with [1251]iodocyan~pindolol or [1251] iodohydroxybenzylpindolol, IC50 values for 8-adrenergic agonists increased 13-200-fold between 1 and 60 min, whereas antagonists yielded similar ICsO values at the two time points. The binding of antagonists, but not agonists, could be simulated by a computer model based on the law of mass action. In contrast with results in intact S49 cells, crude membrane fractions yielded similar ICSo values for agonists in 1- and 60-min incubations with [1Z51]iodocyanopindolol. Moreover, time-dependent decreases in apparent affinity of the agonist (-)-isoproterenol were observed not only in wild type S49 cells but also in several S49 variants (UNC, cyc-, H21a) having defects in N,, the guanine nucleotide binding protein that couples receptors to activation of adenylate cyclase, and in Kin-, an S49 variant with absent CAMP-dependent protein kinase activity. These results show that &adrenergic receptors of intact 549 cells demonstrate a prominent timedependent decrease in apparent affinity for agonists and that this decrease in affinity does not require cAMP generation, the N, components defective in several S49 variants or CAMP-dependent protein kinase. The rapid conversion of agonist binding from high to low affinity can account for the rapid desensitization of intact cells to catecholamines and probably explains previously reported KD/KaCt discrepancies of intact cells.