Abstract
Cellular levels of G protein-coupled receptor kinase (GRK)3 determine the sensitivity of the α2A/B-adrenoceptor (α2-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 Ca2+-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.
Footnotes
-
This research was supported by American Heart Association, Texas Affiliate Inc., Grant 0555032Y (to D.C.E.). Some of the data in this manuscript were presented: Salim S and Eikenburg DC (2007) Regulation of GRK3 expression via ERK1/2-mediated transcription and Hsp90-mediated transcription and Hsp90-mediated protein stabilization, in Society for Experimental Biology Meeting; 2006 April; San Francisco, CA. ASBMB, Bethesda, MD, and Eikenburg DC and Salim S (2006) Regulation of GRK3 expression via ERK1/2-mediated transcriptional regulation and Hsp90-mediated protein stabilization, in the Keystone Symposium G-protein Coupled Receptors: Evolving Concepts and New Techniques; 2006 Feb; Keystone, CO.
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.106.114835.
-
ABBREVIATIONS: GRK, G protein-coupled receptor kinase; GPCR, G protein-coupled receptor; AR, adrenoceptor; CRF, corticotropin-releasing factor; Hsp, heat shock protein; EPI, epinephrine; ALLN, N-acetyl-l-leucyl-l-leucyl-l-norleucinol; PAGE, polyacrylamide gel electrophoresis; GA, geldanamycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; BCA, bicinchoninic acid.
- Received September 29, 2006.
- Accepted December 18, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|