Effect of activating and inactivating mutations of G S ‐and G i2 ‐alpha protein subunits on growth and differentiation of 3T3‐L1 preadipocytes

Previous investigations have demonstrated that both G s ‐ and the G i ‐family of GTP‐binding proteins are implicated in differentiation of the 3T3‐L1 preadipocyte. In order to further analyze the role of G s α vs. G i2 α, 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‐G s α or the inactivating G226A(H21a)‐G s α point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the G i2 α wild type or the missense mutations R179E‐G i2 α, Q205L‐G i2 α, and G204A(H21a)‐G i2 α. The activating [R201C]G s α‐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]G s α‐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]G i2 α or [Q205L]G i2 α mutants reduced cell doubling time in non‐confluent 3T3‐L1 cell cultures, while [H21a]G i2 α 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]G i2 α or [Q205L]G i2 α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]G i2 α and [Q205L]G i2 α 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]G i2 α and [Q205L]G i2 α mutants. Finally, steady‐state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]G i2 α and [Q205L]G i2 α over differentiated controls. The inactivating [H21a]G i2 α‐mutant obliterated all signs of preadipocyte differentiation. It is concluded that G i2 plays a positive and much more important role than G s 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.