Abstract
[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.
Highlights
[’HIForskolin binds to human platelemt embranes in nists regulate adenylate cyclase by facilitating the association the presence of 5 mM MgClz with a B, of 125 fmol/ of guanine nucleotides to or Ni proteins [1,2].Guanine mg of protein and a K d of 20 nM
A slow linear The ECbOfor the synergistic interactions of forskolin with increase in [‘Hlforskolin binding is observed over a hormones is between 10nM and 100nM, which is substantially period of 60 min when Gpp(NH)p and [‘Hlforskolin lower than forskolin’s EC50 for direct activation of adenylate are added simultaneously to membranes
Gpp(NH)p.In human platelet membranes, agents that binding sites have a K d of about 20 nM which is close to the activate adenylate cyclase via the guanine nucleotide EC50 for the synergistic effects of forskolin on thestimulation stimulatory protein (N.) increase the number of bind- of adenylate cyclase by hormone-receptor agonists
Summary
[’HIForskolin binds to human platelemt embranes in nists regulate adenylate cyclase by facilitating the association the presence of 5 mM MgClz with a B,, of 125 fmol/ of guanine nucleotides to or Ni proteins [1,2].Guanine mg of protein and a K d of 20 nM. The increase in tnhuember of [‘Hlforskolin platelets have receptors for PGE, and PGD, that stimulate binding sitesobserved in thepresence of NaF is unaf- adenylate cyclase through the N. protein [14,15,16,17,18,19,20]. The bindingof [3H]forskolin concentration of forskolin that does not stimulate adenylate to membranes that are preincubated with Gpp(NH)p cyclase in human platelets can elevate the maximal rate of for 120min or assayed inthe presence of PGEl reaches CAMPaccumulation in thepresence of PGE, and PGD, [11].
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