G protein-coupled receptors (GPCRs) receive numerous extracellular stimuli and transmit intracellular signals through heterotrimeric G proteins that consist of a Gα subunit and a Gβγ dimer. Upon receptor activation, the Gα subunit dissociates from or reorientates with the Gβγ dimer, enabling both entities to regulate disparate and overlapping signaling pathways. To elucidate the signals solely generated by the Gβγ dimer upon the activation of GPCR, signals stemming from the Gα must be removed first. Since the Gβγ-induced signals were frequently observed upon activation of Gi-coupled receptors, it would be tremendously valuable to engineer the Gα_i1 subunit into an effector-deficient mutant, while leaving its other functions intact. The canonical effector of Gα_i1 is the adenylyl cyclase (...[ Read more ]

G protein-coupled receptors (GPCRs) receive numerous extracellular stimuli and transmit intracellular signals through heterotrimeric G proteins that consist of a Gα subunit and a Gβγ dimer. Upon receptor activation, the Gα subunit dissociates from or reorientates with the Gβγ dimer, enabling both entities to regulate disparate and overlapping signaling pathways. To elucidate the signals solely generated by the Gβγ dimer upon the activation of GPCR, signals stemming from the Gα must be removed first. Since the Gβγ-induced signals were frequently observed upon activation of Gi-coupled receptors, it would be tremendously valuable to engineer the Gα_i1 subunit into an effector-deficient mutant, while leaving its other functions intact. The canonical effector of Gα_i1 is the adenylyl cyclase (AC; esp. AC1, AC5, and AC6 isoforms) which regulates the intracellular cAMP levels. Therefore, my primary focus is on identifying the AC-interacting sites and regions in Gα_i that contribute to its AC inhibitory function.

As the precise AC-interacting domains on Gα_i members remain undefined, four sets of Gα_i1 mutants were constructed with the following considerations: (i) residues cognate to the AC-activating domains on Gα_s members, which are known to interact with one of the conserved cytosolic catalytic domains (C2 domain) on AC; (ii) residues in switch II region, commonly employed by Gα for effector interaction; (iii) residues conserved among AC-inhibiting Gα subunits; (iv) protein-protein docking analysis between a resolved crystal structure of activated Gα_i1 (PDB: 1GIL) and a computationally simulated structure of AC5 isoform (AlphaFold: ADCY5). By means of cAMP functional assays, the residue I212 and the loop region NKW (256-258) in Gα_i1 are shown to be involved in AC inhibition. One of the constructs - the Chi1- NKW mutant has successfully become effector-deficient and will be further tested before proceeding to detect Gβγ signals. Interestingly, the inhibitory profiles of the Gα_i1 mutants induced by the two commonly employed constitutive active mutations (R178C and Q204L) differ significantly. One of the putative effector-interacting mutations on the α4 helix and α4/β6 loop region (i.e. Chi1) has impaired the GDP/GTP exchange rate of Gα_i1. Also, the Chi1 (RC) mutant is sensitive to the negative regulation by RGS proteins.