Role of 90-kDa Heat Shock Protein (Hsp 90) and Protein Degradation in Regulating Neuronal Levels of G Protein-Coupled Receptor Kinase 3

Abstract

Cellular levels of G protein-coupled receptor kinase (GRK)3 determine the sensitivity of the ␣ 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 90-kDa heat shock protein (Hsp90) and GRK3; Hsp90 reportedly affects the maturation and stability of GRK2. In unstimulated cells, GRK3 coimmunoprecipitates with Hsp90, suggesting a physical interaction. Moreover, when GRK3 protein expression was increased by 24-h 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, proteasome-mediated, 70% decrease in GRK3 levels within 24 h. 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 percentage of increase in GRK3 by EPI was not significantly different in the absence versus presence of GA (141 Ϯ 41 versus 94 Ϯ 12%). Finally, we examined the influence of Ca 2ϩ -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.

Increased G protein-coupled receptor kinase (GRK) expression in the anterior cingulate cortex in schizophrenia

“…Results from test Western blots gave a prominent band at the predicted molecular weight for each isoform based on antibody data sheets. Representative bands from our Western blots are consistent with previous reports from the literature (Ahmed et al, 2008a; Bezard et al, 2005; Bychkov et al, 2011a; Bychkov et al, 2012; Bychkov et al, 2011c; Bychkov et al, 2008a; Ertley et al, 2007; Salim and Eikenburg, 2007; Zhang et al, 2014). …”

Pan-histone deacetylase inhibitor panobinostat depletes CXCR4 levels and signaling and exerts synergistic antimyeloid activity in combination with CXCR4 antagonists

“…Chaperone association of CXCR4 with hsp90 in AML cells was further confirmed by the observation that treatment with AUY922 also disrupted the binding of CXCR4 to hsp90 and depleted CXCR4 levels. Consistent with the previous report that GRKs, including GRK3, are also chaperoned by hsp90, 43,44 findings presented here demonstrate that PS and AUY922 treatment also depletes GRK3 levels and inhibits GRK3 binding to hsp90 in AML cells. In addition, treatment with PS also lowered the levels of other signaling hsp90 client oncoproteins in AML cells, including AKT and c-RAF, which are activated by CXCL12 binding and activation of CXCR4.…”

Low concentration of GA activates a preconditioning response in HepG2 cells during oxidative stress—roles of Hsp90 and vimentin

“…Zou et al (1998) have shown that GA also disrupts a complex consisting of Hsp90 and the heat shock transcription factor HSF1 and triggers the activation of a heat shock response in mammalian cells. Low GA treatment might therefore have a binary function, one to elevate the level of Hsp90 similar to preconditioning by the heat shock response mentioned above, and the other to block the binding of substrates with the association of Hsp90 which may result in reducing the degradation of stress-induced misfolded proteins (Salim and Eikenburg 2007;Young et al 2004).…”