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The C type natriuretic peptide receptor associates with AHNAK in human aortic vascular smooth muscle cells
Author(s) -
Alli Abdel A,
Gower William R
Publication year - 2008
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.22.1_supplement.1046.5
Subject(s) - immunoprecipitation , colocalization , gene isoform , natriuretic peptide , receptor , biology , microbiology and biotechnology , peptide , immunohistochemistry , vascular smooth muscle , cell culture , biochemistry , medicine , endocrinology , gene , genetics , immunology , smooth muscle , heart failure
We recently identified AHNAK as a binding partner for the C type natriuretic peptide receptor (NPR‐C) in normal rat gastric mucosa (RGM1) cells. To investigate the role of AHNAK in NPR‐C function in different cell types and species we examined the expression and interaction of both proteins in multiple cell lines. Antibodies to NPR subtypes and AHNAK domains were used to examine expression by immunoblotting, colocalization by immunohistochemistry, and interaction by immunoprecipitation in multiple mammalian cell lines. Mass spectrometry was used to confirm the identity of the proteins from the immunoprecipitation studies. GST fusion proteins of the N‐terminal, internal repeating, and C‐terminal domains of AHNAK were purified and used to perform GST pulldown assays to verify the association of the two proteins and determine specificity. Coding sequences of human NPR‐C and AHNAK were subcloned into mammalian two hybrid expression vectors to ascertain the interaction in situ. NPR‐C was found to be the most abundantly expressed NPR subtype and AHNAK was found to also be expressed in human aortic vascular smooth muscle cells (AoVSMCs). NPR‐C and AHNAK colocalized to the plasma membrane of AoVSMCs. NPR‐C bound to AHNAK isoform 1 in vitro. The interaction was attributed to a single AHNAK domain. This research was supported by an American Heart Association Predoctoral fellowship and a Veterans Affairs Merit grant.