Protein kinase C-mediated gonadotropin-releasing hormone receptor sequestration is associated with uncoupling of phosphoinositide hydrolysis.
Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release by a Ca2+-dependent mechanism involving receptor-mediated phosphoinositide hydrolysis. Previous studies indicate that activation of pituitary protein kinase C (PKC), while not required for acute gonadotropin release in response to GnRH, is likely involved in the chronic regulation of gonadotrope responsiveness. Studies from our laboratory have shown that activation of PKC by phorbol esters produces both the uncoupling of GnRH-stimulated phosphoinositide hydrolysis and the selective enhancement of GnRH agonist binding in pituitary cell cultures. In the present work, we have examined the possibility that these processes are related in mechanism. Dissociation of bound agonist radioligand at 23 degrees C was found to be reduced in the presence of phorbol esters, and ligand bound in the presence of phorbol ester was resistant to displacement by competing ligands at 4 degrees C. However, agonist bound in the presence of phorbol ester was dissociable by subsequently washing cells at pH 3. Receptor photoaffinity labeling studies confirmed that agonist association with membrane component(s) identified as the GnRH receptor was increased in the presence of phorbol ester. These results suggest that, in the presence of a phorbol ester PKC activator, agonist-occupied GnRH receptors remain at the cell surface, but are sequestered in some manner. In other experiments, cell preloaded with [3H]inositol were treated with GnRH agonist ligand and phorbol ester at 4 degrees C to form a pool of sequestered, agonist-occupied receptors, and then displaceable (nonsequestered) agonist was removed by incubation with antagonist ligand. After addition of LiCl and warming to 37 degrees C, [3H]inositol phosphate production (an index of phosphoinositide hydrolysis) in phorbol ester-treated cells was reduced to 67% of vehicle control, although residual specific agonist binding had been increased to greater than 300% of control. The appearance of sequestered receptors and inhibition of [3H]inositol phosphate production had similar phorbol ester concentration dependencies. These results suggest that the same agonist-occupied GnRH receptors sequestered as a result of PKC activation also are preferentially uncoupled from phosphoinositide hydrolysis.
Huckle
W R
WR
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242-1109.
Hawes
B E
BE
Conn
P M
PM
D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D005260:Female; D006868:Hydrolysis; D007700:Kinetics; D007986:Luteinizing Hormone / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011966:Receptors, LHRH / Q000187:drug effects / Q000378:metabolism*
D010716:Phosphatidylinositols; D011966:Receptors, LHRH; D015240:Phorbol 12,13-Dibutyrate; D007986:Luteinizing Hormone; D011493:Protein Kinase C