Initial Events in Phorbol Ester Binding to GH4C1Rat Pituitary Cells

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Abstract
Phorbol ester receptors in GH4C1 rat pituitary cells have been studied using [3H]phorbol 12,13-dibutyrate (PDBu). Binding to intact cells was of higher affinity at 37.degree. C than at 4.degree. C (Kd, .apprx. 8 or .apprx. 34 nM, respectively). The dissociation rate (measured at 4.degree. C) of PDBu from cells equilibrated at 37.degree. C was slower than that from cells equilibrated at 4 C, indicating the temperature-dependent formation of a high affinity (more slowly dissociating) complex in intact cells. Binding to isolated membrane preparations was of low affinity (Kd, .apprx. 30 nM) at 4 C and 37 C; however, binding affinity was increased by including phosphatidylserine in the standard assay mixture (Kd, .apprx. 4 nM). Membrane binding did not account for all of the PDBu binding associated with intact cells. A cytosolic receptor dependent on phospholipids for binding was also studied. The sum of the receptors recovered in the cytosol plus membrane fractions of cell homogenates was very similar to the amount measured in intact cells. This indicates that the cytosolic receptor contributes significantly to the total receptors measured in the intact cell assay. Treatment of cultures with phorbol esters leads to a decrease in receptor concentration in the cytosol and a corresponding increase in receptor density in the membrane fraction. This redistribution indicates that phorbol esters stabilize the membrane association of the cytosolic receptor. The Kd for binding of PDBu to the cytosolic receptor (1.7 nM) and the ED50 for in vitro activation of protein kinase C (4.3 nM) were similar. This result is consistent with this enzyme being a mediator of certain phorbol ester-directed responses in GH4C1 cells.

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