Identification and localization of G-proteins in the clonal adipocyte cell lines HGFu and Ob17

HGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn2+. Gs alpha (largely the long isoform), Gi alpha 2, and G beta were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP-ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. Gs alpha showed vesicular cytoplasmic and nuclear staining; Gi alpha 2 colocalized with actin stress fibers and disruption of these structures altered the distribution of Gi alpha 2; beta subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the Gi alpha 2 and beta fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G-protein subunits, although reorganization of some of these subunits does accompany actin reorganization.