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: Gs alpha, Gi alpha 1, Gi alpha 2, Go alpha, Gz alpha, and G beta.

Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 mumol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits.

Concomitant with an enhanced expression of mu-opioid binding sites, the levels of the inhibitory G proteins Gi alpha 1 and Gi alpha 2 were found to be significantly increased.

This coordinate up-regulation is accompanied by functional changes in mu-opioid receptor-stimulated low-Km GTPase, mu-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5'-(beta gamma-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 prostaglandin E1 (PGE1) receptors and Gs alpha, 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 Gs alpha 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.

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