Regulation of thyrotropin-releasing hormone receptor binding and phospholipase C activation by a single GTP-binding protein.

D L Aub,M E Gosse,T E Cote

doi:10.1016/s0021-9258(18)47964-1

Abstract

In a crude membrane preparation of rat 7315c cells, GTP was found to enhance thyrotropin-releasing hormone- (TRH) stimulated inositol triphosphate (IP3) formation with a potency of 0.97 +/- 0.1 microM. TRH stimulation of IP3 formation was inhibited by high GDP concentrations. Neither nucleotide had any effect in the absence of TRH. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) stimulated IP3 formation in the absence of TRH; the apparent affinity of GTP gamma S was 0.16 +/- 0.05 microM. GTP blocked GTP gamma S stimulation of IP3 formation in a concentration-dependent manner. The apparent affinity of GTP for the site of action shared by GTP gamma S was calculated to be 0.98 +/- 0.3 microM. TRH was able to reverse inhibition of GTP gamma S-stimulated IP3 formation by GTP but could not reverse inhibition by GDP. A lag in the rate of IP3 formation in response to GTP gamma S was abolished by addition of TRH. These data support the proposal that activation of the TRH receptor enhances turnover of guanine nucleotides at the binding protein coupling the receptor to phospholipase C. In addition, GTP gamma S diminished high affinity [3H]Me-TRH binding. The potency of GTP gamma S at decreasing [3H]Me-TRH binding was 0.092 +/- 0.03 microM. GTP gamma S (0.1 microM) decreased the affinity of the TRH receptor for [3H]Me-TRH from 2 to 100 nM. Maximally effective concentrations of GTP gamma S, Gpp(NH)p, GTP, and GDP decreased specific [3H]Me-TRH binding by 80%. Pretreatment of cells with pertussis toxin (30 ng/ml for 24 h) failed to affect TRH receptor affinity or the potency or efficacy of GTP gamma S in diminishing [3H]Me-TRH binding, supporting the identification of Gp (a GTP-binding protein associated with phospholipase C and Ca2+-mobilizing receptors) as distinct from Gi (an inhibitory GTP-binding protein). In contrast to its lack of effect on TRH receptor binding, 3-h pertussis toxin treatment decreased agonist affinity of the mu-opiate receptor and abolished the ability of GTP gamma S to shift the affinity of the mu-opiate receptor for its agonist. The affinities calculated for GTP, GDP, GTP gamma S, and Gpp (NH)p for the G-protein regulating receptor affinity and IP3 formation are nearly identical for each guanine nucleotide tested, suggesting the same G-protein regulates both activities.

Highlights

In a crude membrane preparation of rat 7 3 1 5 ~cells, GTP-binding proteins (G-proteins)’ play a crucial role in GTP wasfound to enhance thyrotropin-releasing hor- coupling numerous hormone receptors to adenylacteyclase as mone- (TRH)stimulated inositol triphosphat(eIps)for- well asphotoreceptorstocGMPphosphodiesterase [1,2,3,4,5]
Proposal that activationof the TRH receptor enhances Pertussis toxin has beenshown to ADP-ribosylate the inhibturnover of guanine nucleotidesat the binding protein itory G-protein (Gi) associated with regulation of adenylate coupling the receptor to phospholipase C
Maximal IP3 formation in response to TRH was achieved only in the presence of exogenous GTP; whenhigh concentrations of GDP were added to the cell lysate, TRH was completely devoid of ability to stimulate phospholipase C

Summary

THEJOURNAOLF BIOLOGICACLHEMISTRY

Val. 262, No 20, Iesue of July 15,ppP. r9i5n2t1e-d9i5n28U.1.S9.8A7. Regulation of Thyrotropin-releasing Hormone Receptor Bindingand Phospholipase C Activation by a Single GTP-binding Protein*. Membrane samples (n= 3) were incubated for 30 min at 37 "C to achieve equilibrium binding (data found that while only the nonhydrolyzable analogs were able not shown) Both TRH andp-opiate receptor binding was linear over to directly stimulate phospholipase C activity, GTP and GDP the range of protein concentrations used These data suggested that activation of the TRH receptor may enhance guanine nucleotide turnshown). Ten p~ GTPyS stimulated a 225% increase in IPSlevels; the EC50 of GTP in blocking the stimulatory effect of GTPyS

RESULTS

GTPYS GTP

PT TREATED MEMBRANES

Nonhydrolyzable GTP analogs are believed to interact with

Findings

The remarkable agreement in the affinities of the various