An extracellular loop 2 mutation P198A prevents the action of the antagonist RC3095 at the gastrin releasing peptide BB2 receptor

L. F. Medeiros1, D. Nesheva1, H. Verli2, R. Roesler3, N. D. Holliday1. 1School of Life Sciences, University of Nottingham, Nottingham, UNITED KINGDOM, 2Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, BRAZIL, 3Department of Pharmacology, Universidade Federal do Rio Grande do Sul, Porto Alegre, BRAZIL.

Introduction. Antagonists for Gastrin releasing peptide (GRP) receptor BB2 have therapeutic potential, for example in analgesia and prevention of tumour proliferation (1,2). Previously, the extracellular loop 2 mutation P198A has been reported to decrease binding affinity for the BB2 agonist peptide bombesin (3). Here we explore the role of Pro198 in the action of GRP, bombesin and the antagonist RC3095 ([D-Tpi6,Leu13y(CH2-NH)-Leu14]bombesin6-14]) (4) at the BB2 receptor.

Methods. HEK293 cells stably expressed SNAP-tagged human BB2 receptor (HEK BB2) or P198A mutant and fluo4 calcium mobilisation measurements were performed as previously described (5). Briefly cells on 96 well imaging plates were loaded using DMEM containing 1.5µM Fluo-4 and 2.5mM probenecid. Antagonist incubations (5 min, 37°C) and agonist treatments were in HEPES buffered saline solution / 0.1% BSA / probenecid, with fluo4 fluorescence was measured on an MDC Flexstation. Concentration response curves were constructed from pooled data using peak agonist responses (Graphpad Prism v7). Pooled pEC50 and Rmax values are mean±s.e.m. from 5-7 experiments, with differences between data groups tested by one way ANOVA followed by Dunnett’s post test.

Results. GRP and bombesin stimulated calcium mobilisation in HEK BB2 cells with pEC50 values of 9.59±0.19 and 10.30±0.22 respectively, and similar Rmax (2.86±0.19 fold over basal for GRP and 2.96±0.15 for bombesin). There were small decreases (P<0.05) in agonist potency for HEK BB2 P198A calcium responses (GRP pEC50 8.92±0.18, bombesin pEC50 9.63±0.18), with Rmax values unaffected. RC3095 was a non-surmountable antagonist of GRP calcium responses in HEK BB2 cells, but in contrast this antagonist was without significant effect on GRP responses in HEK BB2 P198A cells (Figure 1).

Conclusions. These data confirm the modest effect of P198A on agonist activation of the BB2 receptor (3), and for the first time identify its critical role in the action of RC3095 as a BB2 receptor antagonist. These inhibitory effects might be attributable to a direct influence on the ligand binding site, or more global effects on BB2 extracellular loop 2 structure.

Supported by a Brazilian CAPES fellowship (#041/2014, LFM), and a Wellcome Trust Vacationship Scholarship (DN).

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