Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase

Anne P Beigneux; Peter Gin; Brandon S. J Davies; Michael M Weinstein; André Bensadoun; Loren G Fong; Stephen G Young

doi:10.1074/jbc.M109.046391

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Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase

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Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase

Anne P Beigneux, Peter Gin, Brandon S. J Davies, Michael M Weinstein, André Bensadoun, Loren G Fong and Stephen G Young

The Journal of biological chemistry, Vol.284(44), pp.30240-30247

10/30/2009

DOI: 10.1074/jbc.M109.046391

PMCID: PMC2781579

PMID: 19726683

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Abstract

GPIHBP1, a glycosylphosphatidylinositol-anchored endothelial cell protein of the lymphocyte antigen 6 (Ly6) family, binds lipoprotein lipase (LPL) avidly and is required for the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 contains two key structural motifs, an acidic domain and an Ly6 motif (a three-fingered domain specified by 10 cysteines). The acidic domain is required for LPL binding, but the importance of the Ly6 domain is less clear. To explore that issue, we transfected cells with a wild-type GPIHBP1 expression vector or mutant GPIHBP1 vectors in which specific cysteines in the Ly6 domain were changed to alanine. The mutant GPIHBP1 proteins reached the cell surface, as judged by antibody binding studies and by the ability of a phosphatidylinositol-specific phospholipase C to release these proteins from the cell surface. However, cells expressing the cysteine mutants could not bind LPL. The acidic domain of the cysteine mutants appeared to remain accessible, as judged by binding studies with an antibody against the acidic domain. We also developed a cell-free assay of LPL binding. We created a rat monoclonal antibody against the carboxyl terminus of mouse GPIHBP1 and used that antibody to coat agarose beads. We then tested the ability of soluble forms of GPIHBP1 that had been immobilized on monoclonal antibody-coated beads to bind LPL. In this assay, wild-type soluble GPIHBP1 bound LPL avidly, but the cysteine mutants did not. Thus, our studies suggest that a structurally intact Ly6 domain (in addition to the acidic domain) is essential for LPL binding.

Lipids and Lipoproteins

Metabolism, Regulation, and Signaling

Details

Title: Subtitle: Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase
Creators: Anne P Beigneux - From the Departments of
Peter Gin - From the Departments of
Brandon S. J Davies - From the Departments of
Michael M Weinstein - From the Departments of
André Bensadoun - the
Loren G Fong - From the Departments of
Stephen G Young - From the Departments of
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.284(44), pp.30240-30247
DOI: 10.1074/jbc.M109.046391
PMID: 19726683
PMCID: PMC2781579
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; 9650 Rockville Pike, Bethesda, MD 20814, U.S.A
Grant note: R01 HL094732; P01 HL090553; R01 HL087228 / National Institutes of Health
Language: English
Date published: 10/30/2009
Academic Unit: Fraternal Order of Eagles Diabetes Research Center; Biochemistry and Molecular Biology
Record Identifier: 9984024504302771

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