Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different conformations of its J-domain

Abstract

Background and purpose: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF(1)), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF(1) by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF(1) states for Gs and Gi coupling are similar or different.

Experimental approach: We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using the [(35)S]-GTPgammaS binding stimulation assay.

Key results: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism.

Conclusions and implications: The results allow us to extend the two-domain model of CRF(1) activation by assuming that CRF(1) agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF(1).

Figure 1

Effect of the peptide antagonist α-helical CRF(9–41) and the non-peptide antagonist antalarmin on the concentration–response curves for sauvagine-stimulated binding of [³⁵S]-GTPγS to HEK-rCRF₁ cell membranes. To observe Gs- (a, b) and Gi-coupled activity selectively (c, d), cell membranes were obtained from HEK-rCRF₁ cells pretreated with 100 ng ml⁻¹ PTX for 24 h and with 0.1 μM sauvagine for 4 h, respectively. About 120 pM [³⁵S]-GTPγS and 5–7 μg of membrane protein were incubated in binding medium (see Methods) with increasing concentrations of sauvagine in the absence and presence of fixed concentrations of α-helical CRF(9–41) (a, c) or antalarmin (b, d) at 25°C for 2 h. The data shown represent a single experiment and are given as means±s.d. from triplicate incubations. They were fitted by nonlinear regression and from the EC₅₀ values obtained, Schild plots were drawn, when competitive antagonism was observed (insets in a, b, and c).

Figure 2

Effect of the peptide antagonist α-helical CRF(9–41) and the non-peptide antagonist antalarmin on the concentration–response curves for urocortin-stimulated binding of [³⁵S]-GTPγS to HEK-rCRF₁ cell membranes selectively expressing Gs (a, b) and Gi-coupled activity (c, d). Experimental conditions were exactly the same as given for sauvagine-stimulated binding in Figure 1.

Figure 3

Maximum stimulation of sauvagine- and urocortin-stimulated [³⁵S]-GTPγS binding to HEK-rCRF₁ cell membranes in the presence of the non-peptide antagonist antalarmin. Data for Gs and Gi activity were estimated from concentration–response curves, as shown in Figures 1 and 2. The data points were normalized with the maximum response in the absence of antagonist taken as 100% (means±s.d.).

Figure 4

Effect of antalarmin on immunoprecipitation of sauvagine-stimulated [³⁵S]-GTPγS-bound G_αi, G_αs and G_αq subunits in membranes obtained from HEK-rCRF₁ cells. The membranes (40 μg) were incubated with 0.4 nM [³⁵S]-GTPγS in the absence (basal) and in the presence of sauvagine (stimulated) with and without 1 μM antalarmin. The G_αi-, G_αs- and G_αq-[³⁵S]-GTPγS complexes were immunoprecipitated using antibodies directed against the G_α subunits, and the [³⁵S]-GTPγS activities in the precipitates were directly counted. The data shown are the responses to sauvagine in the presence of antalarmin as percentage of those in absence of antalarmin. Data are given as mean±s.e. of at least four experiments carried out in triplicate. Statistically significant changes in sauvagine-evoked [³⁵S]-GTPγS binding caused by antalarmin are indicated as ^*P<0.05.