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Proteomic assessment of the binding partners of GRK4γ: an approach to understand the molecular mechanisms of GRK4γ action
Author(s) -
Nguyen Jenny H,
Liu Jamie,
Adams Craig W,
Andresen Bradley T
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1172.10
Subject(s) - hsp90 , ubiquitin , ubiquitin ligase , g protein coupled receptor kinase , g protein coupled receptor , microbiology and biotechnology , biology , receptor , signal transduction , biochemistry , heat shock protein , gene
The molecular mechanism of action of G protein‐coupled receptor (GPCR) kinase 4γ (GRK4γ) remains largely unknown. In an attempt to better understand how GRK4γ recognizes specific GPCRs and its molecular mechanism of action, we utilized a proteomic approach to identify proteins that interact with GRK4γ. This approach produced dozens of potential interactions. Two interesting interactions were Hsp90 and ubiquitin ligase E3C. Hsp90 is known to interact with other GRKs; to validate this interaction with GRK4γ we co‐immunoprecipitate transfected GRK4γ and endogenous Hsp90. Similar to GRK2, Hsp90 and GRK4γ interact and are disrupted by Geldanamycin (GA), an Hsp90 inhibitor. Moreover, GA decreased the expression of endogenous GRK4γ in a dose and time dependent manner. Additionally, bands that resemble ubiquitinated GRK4γ were observed; to investigate this GRK4γ was immunoprecipitated and blotted for ubiquitin in the presence and absence of proteosome inhibitors. The data clearly show that GRK4γ is ubiquitinated. Together these data indicate that there are a number of proteins that interact with GRK4γ and dynamically regulate the expression of GRK4γ, which may contribute to the function of GRK4γ. This work was partially funded by NSF PFI and Beckman Coulter Foundation grants.