Retinoic Acid-Induced Differentiation of Human Neuroblastoma SH-SY5Y Cells Is Associated with Changes in the Abundance of G Proteins

Abstract: Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH- SY5Y cells: Gaα, Giα1, Gjα2, Gcα, Gzα, and Gβ. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 μmol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of μ-opioid binding sites, the levels of the inhibitory G proteins Giα1 and Gjα1 were found to be significantly increased. This coordinate up-reg- ulation is accompanied by functional changes in μ-opioid receptor-stimulated Iow-Km GTPase, μ-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5′-(βγ-imido)triphosphate [Gpp(NH)p; 10 nmol/ L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prosta- glandin E1 (PGE1) receptors and Gsα, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE1 binding sites and PGE1 stimulated adenylate cyclase activity, but significantly reduced amounts of Gzα were found. This down- regulation is paralleled by a decrease in the stimulatory activity of Gzα as assessed in S49 cyc- reconstitution assays. However, the reduction in Gaα levels had no effect on both intrinsic and receptor-independent-activated [Gpp(NH)p or forskolin; 100 μtmol/L each] adenylate cyclase, suggesting that the amount of Gzα is in excess over the functional capacity of adenylate cyclase in SH-SY5Y cell membranes. Additional quantitative changes were found for Gzα, Gcα, and Gβ subunits. In contrast, neuronal differentiation in the presence of 12-O-tetradecanoylphor- bol 13-acetate (16 nmol/L; 6 days) failed to affect G protein abundance. Our results provide evidence for a specific RA effect on the abundance of distinct G protein sub- units in human SH-SY5Y neuroblastoma cells. These alterations might contribute to functional changes in transmembrane signaling pathways associated with RA-in- duced neuronal differentiation of the cells.