Differential sensitivity of agonist- and antagonist-occupied gonadotropin-releasing hormone receptors to protein kinase C activators. A marker for receptor activation

W. R. Huckle, C. A. McArdle, P. M. Conn

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

Gonadotropin-releasing hormone (GnRH) stimulates release of pituitary gonadotropins by activating specific plasma membrane receptors. In the present studies, we have used activators of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) to probe the binding characteristics of agonist- or antagonist-occupied GnRH receptors in intact cell cultures, using a radioligand receptor assay. Specific binding of [125I-Tyr5,D-Ser(tBu)6,Pro9,NHEt]GnRH (Buserelin), a high-affinity GnRH agonist, was increased to 180% of control in the presence of 150 nM phorbol 12-myristate 13-acetate (PMA) or 100 nM phorbol 12,13-dibutyrate (PDB), and to 125% of control in the presence of 200 μM 1,2-dioctanoylglycerol, after 20 min at 23°C. The PMA effects were associated with apparent increases in both binding affinity and number of binding sites. The effects of protein kinase C activators on Buserelin binding were concentration- and time-dependent and were not seen with 4α-PMA or 1,2-dioctanoyl-3-Cl-glycerol, neither of which activate protein kinase C. In contrast, PMA had no measurable effects on specific binding of a GnRH receptor antagonist, Ac[D-pCl-Phe1,2,D-Trp3,125I-Tyr5,D-Lys6,D-Ala10]GnRH. When cell cultures were pretreated with 100 nM PDB in the absence of GnRH and then washed to remove the phorbol ester, no effects of prior protein kinase C activation were detected upon subsequent addition of Buserelin. However, when PDB pretreatment was carried out in the presence of 0.3 μM GnRH, residual enhancement of Buserelin binding, but not antagonist binding, was observed at either 23 or 4°C. The radiolabeled agonist activated, and the antagonist blocked, GnRH receptor-mediated luteinizing hormone release and [3H]inositol phosphate production in cells preloaded with [3H]inositol. These findings suggest that the action of protein kinase C on the GnRH receptor, either direct or indirect, requires the receptor to be in an activated (agonist-occupied) state but does not require receptor internalization. The mechanism of these effects on GnRH agonist binding is not known but may involve sequestration of surface receptors, expression of new receptors, and/or modulation of GnRH receptor affinity.

Original languageEnglish
Pages (from-to)3296-3302
Number of pages7
JournalJournal of Biological Chemistry
Volume263
Issue number7
StatePublished - 1988
Externally publishedYes

Fingerprint

Dive into the research topics of 'Differential sensitivity of agonist- and antagonist-occupied gonadotropin-releasing hormone receptors to protein kinase C activators. A marker for receptor activation'. Together they form a unique fingerprint.

Cite this