Trimeric G proteins are critical components of cellular signal transduction pathways. The a subunit of G_z protein (α_z) is the only pertussis toxin-insensitive member of a-subfamily which is not well characterized. The present study focuses on unraveling the structure-function relationships of α_i with three major emphases: (1) receptor coupling domains, (2) interactions with [beta gamma] complex, and (3) adenylyl cyclase inhibiting domains. Chimeric [aplha] subunits were constructed by replacing portions of α_z sequence with the corresponding regions of other α subunits for identifying various functional domains. Site-directed mutagenesis was applied to mutate particular residues of [alpha][alpha] _z for studying their specific roles. Desired recombinant proteins were co- expressed in mamm...[ Read more ]

Trimeric G proteins are critical components of cellular signal transduction pathways. The a subunit of G_z protein (α_z) is the only pertussis toxin-insensitive member of a-subfamily which is not well characterized. The present study focuses on unraveling the structure-function relationships of α_i with three major emphases: (1) receptor coupling domains, (2) interactions with [beta gamma] complex, and (3) adenylyl cyclase inhibiting domains. Chimeric [aplha] subunits were constructed by replacing portions of α_z sequence with the corresponding regions of other α subunits for identifying various functional domains. Site-directed mutagenesis was applied to mutate particular residues of [alpha][alpha] _z for studying their specific roles. Desired recombinant proteins were co- expressed in mammalian cell lines and the functional alterations of various [alpha][alpha] _z mutants were compared with α_z and other α subunits by monitoring the changes of second messenger productions. The results suggested that both termini of α_zsubunit were critical for receptor coupling, while the N-terminal 40 residues appeared to be more important on specifying receptor coupling. Mutation of a conserved arginine residue remote from receptor interacting domains disrupted the receptor-mediated activation of G proteins in different subfamilies. By examining various N-terminal serine mutants of α_z it was found that the signaling events involving G_z were not disrupted by protein kinase C (PKC)-mediated phosphorylation. Instead, Ser-27 of α_z as well as the corresponding residue of α_t1 was involved in defining the affinity for [beta gamma] complex. Furthermore, loss of both, but not either one, of the PKC-targeted serine residues on the N-terminus of α_z perturbed the constitutive inhibitory action of a GTPase- deficient α_z mutant. Previous studies suggested that the C-terminal 40% of the sequences of the various α subunit contained the effector interacting domains. The adenylyl cyclase inhibiting domains of α_z was studied using [alpha][alpha] _t1/α_z chimeras. One of the essential adenylyl cyclase inhibiting domains was a stretch of 24 residues within the C-terminal portion of [alpha][alpha] _z located at the α4/β6 loop in the putative tertiary structure. The first 212 residues of a, also contained determinants for adenylyl cyclase inhibition that may be missing in α_s/α_z and α_s/[alpha][alpha] _i2 chimeras.