Characterization of the molecular mechanisms of the coupling between intracellular loops of prostacyclin receptor with the C-terminal domain of the Gαs protein in human coronary artery smooth muscle cells

The C-terminal domain of the Gs protein α subunit (Gαs Ct) and the first intracellular loop (iLP1) of prostacyclin receptor (IP) have been predicted to be involved in the receptor signaling mediated through the IP/Gs protein coupling by our previous NMR studies using synthetic peptides. To test whether the results of the peptide studies can be applied to the protein interaction between the IP receptor and the Gs protein in cells, a minigene technique was used to construct cDNAs that encoded either the amino acid residues of the Gαs or that of the individual intracellular loops of the IP receptor. The effects of the minigene-expressed protein fragments on cAMP production mediated by the IP/Gs coupling were evaluated through experiments that co-expressed peptides either through the Gαs Ct or the IP intracellular loops with the IP receptor in HEK293 cells. The first (iLP1) and third (iLP3) IP intracellular loops, as well as the Gαs Ct, which are important to the IP/Gs coupling-mediated signaling, were identified by the significant reduction of cAMP production when the corresponding peptides were expressed in the cells. Furthermore, the cAMP productions were significantly impaired in Gαs-knockout cells co-expressing the IP receptor with the Gαs C-terminal mutants (E392A, L393A and L394A), compared with the Gαs wild type. Blocking of the endogenous IP/Gs coupling by the minigene-expressed peptides of the Gαs CT, iLP1 and iLP3 was further observed in the human coronary artery smooth muscle cells (SMCs). These results indicate that the three residues (E392–L394) of the Gαs protein predicted from NMR peptide studies, and the IP iLP1 and iLP3 play important roles in the Gαs-mediated IP receptor signaling in the cells, which may be a general binding site for the corresponding regions of the other prostanoid receptors that couple to Gs protein.