Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 103-111).

published_or_final_version / Zoology / Master / Master of Philosophy

G protein coupled receptors, G proteins and their downstream effectors adenylyl cyclase (ACs) were thought to transiently interact at the plasma membrane by random collisions following agonist stimulation. However a growing number of studies have suggested that a major revision of this paradigm was necessary to account for signal transduction specificity and efficiency. The revised model suggests that signalling proteins are pre-assembled as stable macromolecular complexes together with modulators of their activity prior to receptor activation. How and where these signalling complexes form and the mechanisms governing their assembly and maintenance are not completely understood yet. Initially, we addressed this question by exploring AC2 interaction with beta2-adrenergic receptors (beta2ARs) and heterotrimeric G proteins as parts of a pre-assembled signalling complex. Using a combination of biophysical and biochemical techniques, we showed that AC2 interacts with them before it is trafficked to the cell surface in transfected HEK-293 cells. These interactions are constitutive and do not require stimulation by receptor agonists. Furthermore, the use of dominant-negative Rab/Sar monomeric GTPases and dominant-negative heterotrimeric G protein subunits proved that AC2/beta2AR and AC2/Gbetagamma interactions occurred in the ER as measured using both BRET and co-immunoprecipitation experiments, while interaction of the Galpha subunits with the above complexes occurred at a slightly later stage. Both Galpha and Gbetagamma played a role in stabilizing these complexes. Our data also demonstrated that stimulation of AC was still possible when the complex remained on the inside of the cell but was reduced when the GalphaS/AC2 interaction was blocked, suggesting that the addition of the GalphaS subunit was required to render the nascent complexes functional prior to trafficking to proper sites of action. Next, we tackled the issue of higher order assembly of effectors and G proteins, using two different AC isoforms and GalphaS as a model. We demonstrated that AC2 can form heterodimers with AC5 through direct molecular interaction in unstimulated HEK-293 cells. AC2/5 heterodimerization resulted in a reduced total level of AC2 expression, which affected cellular accumulation of cAMP upon forskolin stimulation. The AC2/5 complex was stable in presence of receptor or forskolin stimulation. We provided evidence that co-expression with GalphaS increased the affinity of AC2 for AC5 as monitored by BRET. In particular, the complex formed by AC2/5 lead to synergistic accumulation of cAMP in presence of GalphaS and forskolin, with respect to either of the parent AC isoforms themselves. Finally, we also showed that this complex can be detected in native tissues, as AC2 and AC5 could be co-immunoprecipiated from lysates of mouse heart. Taken together, we provided evidence for stable formation of signalling complexes involving receptor/G proteins/adenylyl cyclase or G proteins/heterodimeric adenylyl cyclases and that G proteins play a crucial role for their assembly and function.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 77-85).

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 126-137).

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

In an attempt to determine the mechanism by which the tripeptide l-prolyl-l-leucyl-glycine amide (PLG, MIF-I) exerts its antiparkinsonian effect, the action of this substance on various postsynaptic components of striatal dopaminergic nerves was studied. It was shown that injection of rats with MIF-I (1 mg/kg, IP×5, 24 hr intervals) did not alter tyrosine hydroxylase, dopa decarboxylase, choline acetyltransferase and glutamic acid decarboxylase activities in the striatum under the conditions tested. The activities of adenylate cyclase, dopamine-stimulated adenylate cyclase, and guanylate cyclase were not altered in vitro by various concentrations of MIF-I (0.1 to 1000 μM), although VIP and neurotensin had some effect. Also the rate of uptake of 3H-dopamine by rat striatal synaptosomes was unchanged, as was the binding of 3H-dopamine and 3H-spiperone to beef caudate membranes. This series of studies indicates that MIF-I does not act directly on the striatal dopamine postsynaptic receptor under the conditions tested, although it is possible that MIF-I could act indirectly at this or another site in vivo by releasing or activating some other factor.

G-proteins are heterotrimeric complexes composed of α, β, and τ subunits and are involved in coupling receptor and effector functions during signal transduction across plasma membranes. G-proteins Gs and Gi are stimulatory and inhibitory to the catalytic subunit of adenylyl cyclase, respectively. A chimeric G-protein α subunit cDNA was constructed from the complete 5' untranslated region of Gαs52 (the 52 kD α subunit of Gs), the first 356 codons of the rat Gαs52, and the last 36 codons and 428 bp of the 3' untranslated region of the rat Gai2 (the α subunit of Gi2) cDNA. Expression of the chimeric G-protein alpha subunit (Gαs/i(38)) causes a constitutive increase in adenylyl cyclase activity in three different fibroblast cell lines. In turn, the elevated cyclase activity leads to higher levels of basal cyclic AMP and protein kinase A activity. The effect of Gαs/i(38) on cyclase does not seem to be through an inhibiton of Gαi function, but instead appears to be the consequence of direct action on the catalytic subunit, resulting in both a decreased time required for maximal cyclase activation and a greater maximal activation as well. Such alterations are not noted in cells expressing exogenous, wild-type Gαs. This data is based primarily on reconstitution assays using cholate extracts from fibroblast Gαs/i(38) clones and membranes derived from the S49 murine T-cell lymphoma (cyc- variant). Endogenous G-protein steady-state changes were detected by immunoblot analysis, but do not appear to account for the observed phenotypic alterations in Gαs/i(38) expressing clones. Furthermore, the validity of the above findings is unequivocally demonstrated through the use of amethopterin-mediated amplification of the chimeric Gαs/i(38) gene transcript and the consequent activation of adenylyl cyclase.

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