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G‐protein Coupled Receptor Kinase 4 gamma (GRK4γ) Mediated Activation of NF‐κB
Author(s) -
Kanassatega RhyeSamuel,
Andresen Bradley T
Publication year - 2016
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.30.1_supplement.1189.10
Subject(s) - luciferase , nf κb , relb , signal transduction , iκb kinase , kinase , gene isoform , microbiology and biotechnology , chemistry , biology , nfkb1 , biochemistry , cell culture , genetics , transcription factor , gene , transfection
Polymorphisms within GRK4γ strongly associate with hypertension, and recently A142V GRK4γ was implicated in NF‐κB‐mediated expression of the angiotensin type 1 receptor (AT 1 R). Luciferase assays driven by the 2x κB pGL3 vector, nuclear fractionation, and pull‐down assays were utilized to investigate the mechanism via which wild‐type (wt) GRK4γ and single nucleotide polymorphisms (SNPs) R65L, A142V, and A486V activate NF‐κB. Luciferase assays were conducted in growth media and serum‐free media. Only in growth media did the SNPs increase NF‐κB activity; wtGRK4γ decreased NF‐κB activity compared to the vector control. Utilizing kinase‐dead mutants of GRK4γ demonstrated that only A142V GRK4γ‐mediated NF‐κB activity is dependent on the kinase activity. The luciferase assays demonstrate that GRK4γ SNPs act as an amplifier, but do not constitutively activate NF‐κB. Nuclear fraction confirmed the luciferase data and demonstrated that RelB is the NF‐κB isoform activated through the GRK4γ pathway. Pull‐down affinity assays with S‐tagged wtGRK4γ and GRK4γ SNPs showed significantly increased interaction between SNP GRK4γ and NEMO (IKKγ). No interaction between the other IKK isoforms (α, β, ɛ) and wtGRK4γ or GRK4γ SNPs was detected. Currently, the IκBs are being examined to complete the NF‐κB‐mediated signaling pathway that is activated by SNPs of GRK4γ. Collectively, the data indicates that polymorphic GRK4γ amplifies serum‐mediated activation of the canonical NF‐κB pathway through a kinase‐mediated (A142V) and protein‐protein interaction‐mediated (R65L & A486V) mechanism. Further elucidation of these mechanisms may allow for the development of drugs that block polymorphic GRK4γ‐mediated NF‐κB activity and consequently reduce AT 1 R expression and thus blood pressure. Support or Funding Information This work was funded from Internal funds from Western University of Health Sciences and American Indian Graduate Center STEM Student Fellowship.