Characteristics of an adenylate cyclase coupled beta 2-adrenergic receptor in a smooth muscle tumor cell line.

Abstract

We have shown that binding of 3H-dihydroalprenolol ( [3H] DHA) to DDT1 MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [3H]DHA dissociation constants were 0.63 +/- 0.15 nM (n = 6) and 0.83 +/- 0.04 nM (n = 5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 +/- 5,200 sites/cell (n = 6) and for membranes 468 +/- 24 fmoles/mg protein (n = 5). The order of agonist competition for the [3H]-DHA binding site of DDT1 cell membranes was isoproterenol (Ki = 0.20 +/- 0.07 microM) greater than epinephrine (Ki = 0.4 +/- 0.2 microM) greater than norepinephrine (Ki = 66.5 +/- 5.15 microM) consistent with a beta 2-adrenergic receptor interaction. Zinterol, a beta 2-selective antagonist, (Ki = 0.05 +/- 0.01 microM) was 18X more effective than metoprolol, a beta 1-selective antagonist (Ki = 0.9 +/- 0.1 microM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p greater than 0.05) DDT1 cells possess a pure population of beta 2-adrenergic receptors. Finally, we have shown that DDT1 MF-2 cell beta 2-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [3H]DHA binding site.