Abstract
Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3.
Highlights
Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IPS)formation and prolactin release from 7315c tumor cells
This hypothesis has been confirmed by the purification of the following: 1) astimulatory guanine nucleotide-binding protein (N:) associated with receptors stimulating adenylate cyclase (Z), 2) an inhibitory guanine nucleotide-binding protein (Ni) associated with receptors inhibiting adenylate cyclase [3, 4], and 3) transducin, a GTPbinding protein coupling photoreceptors to a cyclic GMP phosphodiesterase in retina ( 5 )
The same laboratory has reported that the pertussis toxin-induced ADP ribosylation of a membrane-bound protein ( M, = 41,000) correlated well with the attenuation of Met-Leu-Phe receptor-mediated arachidonate release and Ca2+mobilization in guinea pig neutrophils [18].These authors and others[17,18,19,20,21] have suggested that Ni or an Ni-like GTP-binding protein is involved in coupling cell-surface receptors to phospholipase C activation and PIP2 breakdown
Summary
Dept. of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. The same laboratory has reported that the pertussis toxin-induced ADP ribosylation of a membrane-bound protein ( M , = 41,000) correlated well with the attenuation of Met-Leu-Phe receptor-mediated arachidonate release and Ca2+mobilization in guinea pig neutrophils [18].These authors and others[17,18,19,20,21] have suggested that Ni or an Ni-like GTP-binding protein is involved in coupling cell-surface receptors to phospholipase C activation and PIP2 breakdown. The abbreviations used are: N., a stimulatory GTP-binding protein; Ni, an inhibitory GTP-binding protein; PIP,, phosphatidylinositol 4,5-bisphosphate; IPS, inositol trisphosphate; TRH, thyrotropin-releasing hormone; GTPyS, 5”guanosine y-thiotriphosphate; Gpp(NH)p, 5’-guanylyl imidodiphosphate; EGTA, [ethylenebis(oxyethylenenitri1o)ltetraacetic acid; HEPES, 4-(2-hydroxyethyl)-lpiperazineethanesulfonicacid; EBSS, Earle’s balanced salt solution; BSA, bovine serum albumin. The findings of the present studysuggest that the TRH receptor on the 7315c tumor cells is coupled to phospholipase C by a guanine nucleotide-binding protein [14]that is distinct from N. or Ni
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have