THESIS
1998
xv, 220 leaves : ill., photos (some col.) ; 30 cm
Abstract
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...[
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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.
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