Saccharomyces cerevisiae: a tool to evaluate the signaling pathways of adhesion-GPCRs.

Adhesion G-protein coupled receptors (aGPCRs) are the second largest family of GPCRs, with 33 homologues in humans. Despite the multitude of roles that they play, the majority of them are still orphans and the signaling pathways involved in their activations barely unknown. The aim of this study is to investigate the constitutive activity of different truncations of the N-terminus of the GPR56, GPR64 and GPR112 receptors that belong to the subfamily VIII of the aGPCRs, in order to shed light on their downstream pathways. Modified strains of Saccharomyces cerevisiae have been used for this study. These strains contain chimeric Gp1a/Gα proteins that are able to couple to a heterologous GPCR and give a response via the pheromone-response pathway. The constitutive activity has been evaluated as the growth of yeast cells in a medium lacking of histidine, since the production of this amino acid (essential for S. cerevisiae) is due to the activation of the receptor. During the initial screen the GPR112-7TM and the GPR112-GPS have been tested in all the strains, coupling different Gα proteins; this was necessary to optimise the assay in itself and to discriminate the strains to use later. In the following part of the study, all the constructs generated by the truncation of the N-termini have been tested in selected yeast strains, based on the initial screen and the information gathered in literature. Intriguingly, the receptors showed different responses among the different strains and a same receptor displayed a different mechanism of activation based on the signaling pathway involved. In fact, the GPR64-ZO1 seems to have interactions with other components on the extracellular region, the GPR56 shows a higher activity when the N-terminal fragment is cleaved out, the GPR112-GAIN (via Gα14) modulates the activity of the receptor, which is fully activated when the N-terminus is removed. Moreover, the GPR64-ZO1 modulates the activity of the receptor positively via Gα14 but negatively via Gα12, the three constructs of the GPR112 show the same level of activation via Gα16, Gα12 and Gα13, but via Gα14 the receptor is fully activated when the N-terminus is removed, the GPR56-7TM has a remarkable activity via Gα12 and Gα13