ISSN:
0730-2312
Keywords:
GTP-binding proteins
;
site-directed mutagenesis
;
stable transfection
;
adenylate cyclase
;
glucose uptake
;
hormone sensitive lipase
;
cell growth
;
cell differentiation
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
Previous investigations have demonstrated that both Gs- and the Gi-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of Gsα vs. Gi2α, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-Gsα or the inactivating G226A(H21a)-Gsα point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the Gi2α wild type or the missense mutations R179E-Gi2α, Q205L-Gi2α, and G204A(H21a)-Gi2α.The activating [R201C]Gsα-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-Ll adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]Gsα-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]Gi2α or [Q205L]Gi2α mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]Gi2α slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]Gi2α or [Q205L]Gi2α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]Gi2α and [Q205L]Gi2α mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]Gi2α and [Q205L]Gi2α mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]Gi2α and [Q205L]Gi2α over differentiated controls. The inactivating [H21a]Gi2α-mutant obliterated all signs of preadipocyte differentiation.It is concluded that Gi2 plays a positive and much more important role than Gs in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process. J. Cell. Biochem. 64:242-257. © 1997 Wiley-Liss, Inc.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
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