Date of Award

12-2002

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Robert E. Gundersen

Second Committee Member

Keith W. Hutchison

Third Committee Member

Dorothy E. Croall

Abstract

In Dictyostelium discoideum organism, the Gα2 subunit of the heterotrimeric G-protein signaling complex plays a pivotal role during the aggregation stage in the Dictyostelium life cycle. The biochemical functions of the G-protein complex include separation of the G-protein coupled receptor from the G-protein subunits, GDP displacement by GTP in the Gα subunit, separation of the Gα monomer from the βγ complex, GTP hydrolysis to GDP, activation of adenylyl cyclase as a downstream effector, and activation of guanylyl cyclase as a separate downstream effector. Upon release from the heterotrimer, the βγ subunits lead to downstream activation of the membrane bound adenylyl cyclase A. The role of the Gα2 subunit is not well defined other than acting as a regulated GTPase to terminate the signal. To further define the role of the Gα2 subunit, a previously constructed library of random mutations in the Gα2 subunit was screened for aggregation negative mutants expressing the Gα 2 protein. Mutants were reconfirmed as chemotaxis negative. One of these mutants, N74D, has an unusual phenotype. After stimulation with the extracellular ligand CAMP, the affinity of the CAMP specific G-protein coupled receptor to this ligand remained high, as in the unactivated state, and the receptor was unable to activate the G-protein. This mutation is in the Gα2 subunit helical'domain, the function of which is not well understood. The N74D mutant provides some insight regarding the mechanistic role of the helical domain in G-protein signaling.

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