Impact of physical and functional interaction of Dopamine D2 and Bradykinin B2 receptors. Preliminar study in human pituitary adenomas

ABELEDO MACHADO A ; ARGERICH BERGADÀ J; VIDAL N; GARCIA C; GIRONACCI MM; CIRUELA ALFÉREZ F ; DIAZ G

Congreso; Reunión Conjunta de Sociedades de Biociencias (SAIC-SAI-AAFE-NANOMED); 2021

Most prolactinomas are effectively treated with dopamine D2 receptors (D2R) agonists. Nevertheless, a subset (~20%) became resistant to the treatment and require extirpation. The molecular mechanisms underlying the escape from dopamine inhibition include alterations in D2R signalling. It has been reported that bradykinin B2 receptor (B2R) is highly expressed in human prolactinomas and can heteromerize with D2R, abolishing Gi signalling of D2R. In the present study, we proposed to assess 1-the physical and 2- the functional interaction of D2R-B2R, and 3- to determine whether those receptors interact in human pituitary adenomas. 1- By using the NanoBiT protein-protein interaction assay (NB-assay): 1a- we validated the formation of BR2-D2R measuring NanoLuciferase (NL) activity in HEK293T cell line transiently transfected with human D2R and B2R fused to one of the two inactive fragments of the split NL. 1b- we studied the coupling of B2R to G proteins in cells transiently transfected with B2R and Gi, Gs, G12/13 or Gq, fused to inactive fragments of the split NL. We found that B2R recruits Gq and Gi after stimulation with B2R specific agonist. 2- We measured the Ca2+ mobilization by Fluo4-NW Assay in HEK293T transiently transfected with B2R alone or in combination with D2R. B2R agonist (100nM) increased intracellular Ca2+ in cells expressing B2R alone or B2R-D2R. D2R agonist or antagonist did not alter B2R signal suggesting that the coupling of protein Gq to B2R is not altered in the B2R-D2R heteromer. 3- B2R-D2R interaction was measured in membrane extracts of human pituitary adenomas by AlphaLisa assay. We found B2R-D2R complexes in prolactinomas and non-secreting tumours, but not in mixed tumours (GH+PRL). We hypothesized that D2R-B2R dimerization is increased in prolactinomas, disturbing D2R signalling by prevailing the D2R-B2R coupling to Gq, promoting resistance to D2R agonists in patients with resistant prolactinomas.