Heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals into intracellular signals by coupling receptors and effectors. Because most of the G protein-coupled receptors are integral proteins, the G proteins need to have a membrane binding capacity to receive signals from the receptors. The alpha subunit of G protein binds tightly to the cytoplasmic face of the plasma membrane without any membrane spanning domain. Fatty acylation of G alpha with myristic acid or palmitic acid, in addition to the beta gamma subunits, plays an important role in anchoring the G alpha subunit. The reversible and dynamic palmitoylation of the alpha subunit of stimulatory G protein (Gs alpha) has been suggested as essential for its membrane attachment. However, in our previous experiments, Gs alpha deleted in the amino terminus containing palmitoylation site, retained its binding capacity when expressed in COS cells. Thus, to evaluate the role of palmitoylation in Gs alpha membrane binding, we constructed and expressed non-palmitoylated mutants of Gs alpha and analyzed their subcellular distributions in COS-1 cells. We found that non-palmitoylated mutants of Gs alpha, C3S- and G2A/C3S Gs alpha, retained their membrane binding capacities in COS-1 cells, demonstrating that palmitoylation is not essential for membrane binding of Gs alpha in COS-1 cells. We also found that the palmitoylation did not change significantly the distribution of Gs alpha in Triton X-114 partition. These results suggest that the palmitoylation of Gs alpha may produce different effects on membrane binding depending on cell types.
Animal ; Blotting, Western ; COS Cells ; Cell Membrane/metabolism ; Detergents/pharmacology ; G-Protein, Stimulatory Gs/metabolism* ; G-Protein, Stimulatory Gs/genetics ; Immunoblotting ; Isoproterenol/metabolism ; Mutagenesis ; Palmitates/metabolism* ; Polyethylene Glycols/pharmacology ; Rats ; Transfection

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.
3',5'-Cyclic-Nucleotide Phosphodiesterase/genetics/metabolism ; Adenylate Cyclase/genetics/metabolism ; Cyclic AMP/*metabolism ; Cyclic AMP-Dependent Protein Kinases/genetics/metabolism ; G-Protein, Stimulatory Gs/genetics/metabolism ; Heterotrimeric GTP-Binding Proteins/genetics/*metabolism ; Human ; Isoenzymes ; Isoproterenol/pharmacology ; Mutation ; Neuroblastoma/*metabolism ; *Signal Transduction ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured