Identification of a PDZ-domain-containing protein that interacts with the scavenger receptor class B type I.
Journal: 2000/July - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
The scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) and cholesterol secretion into bile in the liver. In this study, we identified an SR-BI-associated protein from rat liver membrane extracts by using an affinity chromatography technique. This protein of 523 amino acids contains four PDZ domains and associates with the C terminus of SR-BI by using its N-terminal first PDZ domain. Therefore, we denoted this protein as CLAMP (C-terminal linking and modulating protein). CLAMP was located mostly in the sinusoidal membranes, whereas SR-BI was detected in both sinusoidal and canalicular membranes. After the solubilization of the liver membranes with Triton X-100, SR-BI was immunoprecipitated with anti-CLAMP monoclonal antibody, suggesting the association of these proteins in vivo. By coexpressing SR-BI with CLAMP in Chinese hamster ovary cells, we observed (i) the increase in the expression level of SR-BI, (ii) the reduction in the deacylation rate of the cholesteryl esters taken up from HDL, and (iii) the change in the intracellular distribution of fluorescent lipid 1,1'-dioctadecyl-3,3, 3',3'-tetramethylindocarbocyanine percholate taken up from HDL. Taken together, these data suggest that CLAMP, a four-PDZ-domain-containing protein, is associated with SR-BI in the liver sinusoidal plasma membranes and may modulate the intracellular transport and metabolism of cholesteryl esters taken up from HDL.
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Proc Natl Acad Sci U S A 97(12): 6538-6543

Identification of a PDZ-domain-containing protein that interacts with the scavenger receptor class B type I

Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan; and Laboratories of Cellular Biochemistry and Biomolecular Characterization, RIKEN (Institute of Physical and Chemical Research), Wako-shi, Saitama, 351-0198, Japan
To whom reprint requests should be addressed. E-mail: pj.ca.oykot-u.f.lom@iarah.
Communicated by John A. Glomset, University of Washington, Seattle, WA
Communicated by John A. Glomset, University of Washington, Seattle, WA
Received 1999 May 24; Accepted 2000 Mar 15.

Abstract

The scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) and cholesterol secretion into bile in the liver. In this study, we identified an SR-BI-associated protein from rat liver membrane extracts by using an affinity chromatography technique. This protein of 523 amino acids contains four PDZ domains and associates with the C terminus of SR-BI by using its N-terminal first PDZ domain. Therefore, we denoted this protein as CLAMP (C-terminal linking and modulating protein). CLAMP was located mostly in the sinusoidal membranes, whereas SR-BI was detected in both sinusoidal and canalicular membranes. After the solubilization of the liver membranes with Triton X-100, SR-BI was immunoprecipitated with anti-CLAMP monoclonal antibody, suggesting the association of these proteins in vivo. By coexpressing SR-BI with CLAMP in Chinese hamster ovary cells, we observed (i) the increase in the expression level of SR-BI, (ii) the reduction in the deacylation rate of the cholesteryl esters taken up from HDL, and (iii) the change in the intracellular distribution of fluorescent lipid 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholate taken up from HDL. Taken together, these data suggest that CLAMP, a four-PDZ-domain-containing protein, is associated with SR-BI in the liver sinusoidal plasma membranes and may modulate the intracellular transport and metabolism of cholesteryl esters taken up from HDL.

Abstract

The level of plasma high-density lipoproteins (HDL) is inversely related to the incidence of atherosclerosis and coronary artery disease (1). The protective effect of HDL is thought to be caused by the reverse cholesterol transport from cells in the arterial wall to the liver for catabolism (2). The scavenger receptor class B type I (SR-BI) is the first HDL receptor that has been well defined at the molecular level and shown to be a mediator of selective cholesterol uptake in vitro (3). Adenovirus-mediated overexpression of SR-BI in the liver leads to reduced plasma HDL levels and increased cholesterol secretion into bile (4). Fluiter et al. (5, 6) have also reported additional strong correlative evidence for the importance of SR-BI in selective cholesterol uptake by hepatocytes in vitro and in vivo.

Recently, SR-BI has been shown to be fatty-acylated and to cluster in caveolae-like domains (7); however, the detailed mechanism of selective uptake mediated by SR-BI has not been defined. The process of selective cholesteryl ester (CE) uptake from HDL can be divided into three steps (8). The first step of selective uptake may involve receptor binding, followed by the reversible incorporation of HDL-CE into the plasma membrane pool and the subsequent transfer of the lipid to an inaccessible pool. It is uncertain whether HDL-CE uptake is achieved by SR-BI itself or whether other cellular components are involved in this process.

SR-BI is a member of the CD36 superfamily, whose members have been proposed to have similar membrane topologies (9). Topologic studies of CD36 in conjugation with sequence analysis indicate that SR-BI has two transmembrane domains that sit adjacent to relatively short cytoplasmic N-terminal (8 residues) and cytoplasmic C-terminal (45 residues) domains. SR-BI and CD36 share significant sequence homology throughout their entire extracellular loop domains; however, the cytoplasmic C-terminal domains were quite different from each other in their primary sequences. Therefore, we assumed that the cytoplasmic C-terminal domain (C45) of SR-BI is important for specific functions of the receptor or its regulations.

In this study, we used recombinant C45-glutathione S-transferase (GST) fusion protein to identify proteins that associate with the cytosolic domain of SR-BI from rat liver membrane extracts. Herein, we describe the isolation and characterization of a PDZ-domain-containing protein that interacts with the C terminus of SR-BI and name this protein CLAMP (C-terminal linking and modulating protein).

The yeast two-hybrid system was used to test the domains of CLAMP required for interaction with SR-BI as described in Materials and Methods. Positive selection on His plates and the β-galactosidase (β-Gal) activity of each construct are indicated.

Acknowledgments

We thank Dr. Yoshimi Takai (Osaka University Medical School, Osaka) for advice on the GST fusion-protein affinity chromatography and Dr. Kazuo Kobayashi (Osaka University) for technical assistance on the preparation of rat liver sinusoidal and canalicular membranes. M.I. is a Special Postdoctoral Researcher.

Acknowledgments

Abbreviations

SR-BIscavenger receptor class B type I
HDLhigh-density lipoprotein
UCunesterified cholesterol
CEcholesteryl ester
CHOChinese hamster ovary
DiI1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholate
GSTglutathione S-transferase
Abbreviations

Footnotes

Data deposition: The nucleotide sequence reported in this paper has been deposited in the GenBank database (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF116896","term_id":"8439548"}}AF116896).

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.100114397.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.100114397

Footnotes

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