Role of 90-kDa heat shock protein (Hsp 90) and protein degradation in regulating neuronal levels of G protein-coupled receptor kinase 3.
Cellular levels of G protein-coupled receptor kinase (GRK)3 determine the sensitivity of the alpha(2A/B)-adrenoceptor (alpha(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 +/- 41 versus 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.
Salim
Samina
S
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204-5037, USA.
Eikenburg
Douglas C
DC
D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism; D045744:Cell Line, Tumor; D004795:Enzyme Stability; D054770:G-Protein-Coupled Receptor Kinase 3; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D015759:Ionomycin / Q000494:pharmacology; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D009474:Neurons / Q000201:enzymology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation; D051552:beta-Adrenergic Receptor Kinases / Q000096:biosynthesis*
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D015759:Ionomycin; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; D051552:beta-Adrenergic Receptor Kinases; D002154:Calpain; C001277:geldanamycin