Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain

Abstract

Background and purpose: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands.

Experimental approach: We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays.

Key results: Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did.

Conclusions and implications: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.

Figure 1

Homologous competition for receptor binding of [¹²⁵I]-Tyr-sauvagine by its unlabelled form I-Tyr-sauvagine in HEK-rCRF₁ cell membranes for different coupling states of the receptor. To obtain the receptor in the states (i) Gs-/Gi-coupled/uncoupled, (ii) Gs-coupled/uncoupled and (iii) Gi-coupled/uncoupled, membranes were prepared from HEK-rCRF₁ cells (i) untreated, (ii) treated with 100 ng ml⁻¹ Pertussis toxin for 24 h and (iii) treated with 0.1 μM sauvagine for 4 h. To obtain the totally uncoupled state (iv), 30 μM GTPγS was added to the incubations using membranes from untreated cells. The membranes (6 μg protein) were incubated with 12 pM [¹²⁵I]-Tyr-sauvagine and increasing concentrations of unlabelled I-Tyr-sauvagine at 25°C for 2 h in the binding medium also used for the GTPγS assay. Data points were normalized with the maximum and non-specific binding taken as 100 and 0%, respectively, and represent the mean±s.d. of triplicates. Curves were fitted according to a one-site (dotted lines, valid for states iii and iv) or two-site competition model (solid lines, valid for states i and ii). The inset gives an example showing the original data for the states ii and iii, as defined above. HEK-rCRF₁, HEK293 cells stably transfected with rCRF₁.

Figure 2

Screening of monosubstituted Bpa-Ucn I analogues for receptor binding affinity and maximum stimulation of G-protein activity at HEK-rCRF₁ cell membranes obtained from cells expressing Gs- as well as Gi-protein activity. (a) From competition binding curves using 100 pM [¹²⁵I]-Tyr-sauvagine, fitted according to a one-site model, K_i values were obtained. These represent the resultant of the receptor in all its different states (uncoupled and coupled to Gs/Gi). When more than one screening experiment was performed, data are given as mean±s.e. (b) The sum of Gs and Gi activity was determined by measuring the stimulation of [³⁵S]-GTPγS binding by 1 μM peptides and related to the maximum activity exhibited by 1 μM Ucn. Data are expressed as mean±s.e. of at least three independent experiments, each with triplicate incubations. HEK-rCRF₁, HEK293 cells stably transfected with rCRF₁.

Figure 3

Influence of Bpa(7)-Ucn I on the G-protein activity, compared with that of Ucn I and Bpa(5)-Ucn I. Using the [³⁵S]-GTPγS binding stimulation assay, the activation of G-protein by the three peptides was determined in membranes obtained from HEK-rCRF₁ cells manipulated to separately express Gs or Gi activity (see Methods). Data (mean±s.d. of triplicates) are expressed as percentage of the maximum activity exhibited by Ucn I. HEK-rCRF₁, HEK293 cells stably transfected with rCRF₁.

Figure 4

Competition of Bpa(7)-Ucn I, Ucn I, and Bpa(5)-Ucn I for the binding of 12 pM [¹²⁵I]-Tyr-sauvagine in membranes obtained from untreated HEK-rCRF₁ cells (receptor uncoupled and coupled to Gs/Gi). Data points of membrane-bound activities represent the mean±s.d. of triplicates and were fitted according to a one-site (valid only for Bpa(7)-Ucn I) and two-site competition model (valid for Ucn I and Bpa(5)-Ucn I). HEK-rCRF₁, HEK293 cells stably transfected with rCRF₁.

Figure 5

Influence of the Gs-selective Bpa(7)-Ucn I at fixed concentrations on the stimulation by sauvagine of the [³⁵S]-GTPγS binding in HEK-rCRF₁ cell membranes expressing selectively Gi activity. Data points represent the mean±s.d. of triplicates and were fitted by nonlinear regression. From the EC₅₀ values obtained, a Schild plot for competitive antagonism was drawn (inset). HEK-rCRF₁, HEK293 cells stably transfected with rCRF₁.

Figure 6

Concentration–response curves for the intracellular cAMP accumulation in HEK-rCRF₁ cells evoked by I-Tyr-sauvagine and the Gs-selective ligands Bpa(7)-Ucn I and Nal(9)-Ucn I. HEK-rCRF₁ cells were stimulated by the peptides for 30 min. The cellular cAMP values were determined by radioimmunoassay and are given (mean±s.d. of triplicates) as percentage of the value obtained by stimulation by 10 μM forskolin. The curves were fitted by non-linear regression, for I-Tyr-sauvagine, separately for the stimulating and inhibiting portion of the bell-shaped curve. HEK-rCRF₁, HEK293 cells stably transfected with rCRF₁.