Identification of coupling specificity between somatostatin receptor 5 (SST5) and G proteins by a bioluminescence resonance energy transfer (BRET) technique: the role of GoA protein
E. Peverelli1, G. Mantovani1, E. Vitali1, M. Busnelli2, B. Chini2, A. Lania1,3, P. Beck-Peccoz1 & A. Spada1
In this study we employed a novel bioluminescence resonance energy transfer (BRET) biosensor to study the coupling specificity of somatostatin receptor 5 (SST5) and its naturally occurring mutant R240W in living cells. Our previous data demonstrated that SST5 carrying the R240W mutation as well as other mutations in the third intracellular loop maintained the ability to inhibit intracellular cAMP levels similarly to the wild-type but failed to mediate the inhibition of intracellular calcium levels, GH release and cell proliferation, suggesting that different regions of SST5 are required for the activation of different signaling pathways. The BRET biosensor may monitor the activation of G proteins in response to SST5 activation by specific agonist BIM23206. The energy transfer occurs within two subunits of the heterotrimeric G protein complex, the energy donor (G alpha-Renilla Luciferase) and the acceptor (G gamma2-GFP10). G protein activation induces a structural rearrangement that increases the distance between the donor and the acceptor with a consequent decrease in the energy transfer. To detect specific G protein activation in living cells, we expressed in HEK293 cells wild-type or mutant SST5 together with different G protein (i1, i2, i3, oA, oB or q). We demonstrated that wild-type SST5 activated Gi (1, 2 and 3) and Go (A and B), whereas R240W SST5 maintained the ability to activate all Gi and GoB, but not GoA. As expected, neither the wild-type nor mutant receptors activated Gq. To investigate the role of GoA in SST5-mediated signal transduction in a pituitary cell model, we cotransfected cultured cells from GH-secreting adenomas with SST5 and a pertussis toxin (PTX)-resistant GoA protein. In PTX-treated cells, GoA restored the ability of BIM23206 to inhibit ERK1/2 phosphorylation and GH secretion. In conclusion, our data first demonstrated the coupling specificity of SST5 and revealed a crucial role for GoA in SST5 signaling in GH-secreting adenomas.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details are unavailable.