Received for publication October 2, 2006.
Revised December 16, 2006.
Accepted for publication December 18, 2006.

Role of Hsp90 and Protein Degradation in Regulating Neuronal Levels of GRK3

Abstract

Cellular levels of GRK3 determine the sensitivity of the alpha_2A/B-adrenoceptor (₂-AR) to agonist-induced down-regulation. Using human neuroblastoma BE(2)-C cells, this study examines how cellular GRK3 levels are affected by several mechanisms reported to influence stability and degradation of other GRKs. We first examined the interaction between the heat shock protein 90 (Hsp90) and GRK3; Hsp90 reportedly affects the maturation and stability of GRK2. In unstimulated cells, GRK3 co-immunoprecipitates (Co-IP) with Hsp90, suggesting a physical interaction. Moreover, when GRK3 protein expression was increased by 24h epinephrine (EPI) treatment, Hsp90 protein expression increased with a similar but slightly delayed time course. To investigate the influence of Hsp90 on GRK3 protein stability, we determined the effect of the Hsp90 inhibitor, geldanamycin (GA) on cellular GRK3 levels. GA eliminated the interaction between Hsp90 with GRK3 and produced a rapid, proteosome-mediated, 70% decrease in GRK3 levels within 24h. To investigate the influence of Hsp90 on up-regulation of GRK3 expression, we examined the effect of GA on EPI-induced up-regulation. GA reduced the absolute increase in GRK3; however, the percent increase in GRK3 by EPI was not significantly different in the absence vs. presence of GA (141±41% vs. 94±12%). Finally, we examined the influence of Ca⁺²-activated proteases on cellular GRK3. Treatment with the calcium ionophore, ionomycin produced a rapid decrease in GRK3 levels that was inhibited by the calpain inhibitor, calpeptin. In conclusion, several mechanisms influence the degradation of GRK3, and therefore have the potential to affect GPCR signaling by regulating GRK3 levels in neurons.

Key words: GRK3, Heat Shock Protein, calpain, degradation, neuron, proteosome