Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling - PubMed (original) (raw)

. 2005 Apr;115(4):969-77.

doi: 10.1172/JCI23858. Epub 2005 Mar 24.

Affiliations

Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling

Chatchawin Assanasen et al. J Clin Invest. 2005 Apr.

Abstract

The binding of HDL to scavenger receptor-BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-beta-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-beta-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-beta-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Cholesterol efflux is required for HDL stimulation of eNOS. (A) eNOS activation was assessed in BAECs by measuring [3H]

L

-arginine to [3H]

L

-citrulline conversion during 15-minute incubations with control buffer (Basal), HDL (50 μg/ml), CD (2%), or cholesterol-loaded CD (Chol-CD, 2%). (B) eNOS activation in BAECs was assessed during 15-minute incubations with control buffer, HDL (5 and 20 μg/ml), or PC-HDL (5 and 20 μg/ml). Values (mean ± SEM) are expressed relative to basal activity, designated as 100%; n = 4. *P < 0.05 versus basal; †P < 0.05 versus no cholesterol. #P < 0.05 versus HDL.

Figure 2

Figure 2

Cholesterol-free Lp2A-I particles activate eNOS. (A) eNOS activation in BAECs was assessed by measuring [3H]

L

-arginine to [3H]

L

-citrulline conversion during 15-minute incubations with control buffer, HDL (20 μg/ml), or Lp2A-I particles with a molar POPC/apoA-I ratio of 80:1 (20 μg/ml). (B) eNOS activation in BAECs was assessed during 15-minute incubations with control buffer, HDL (1, 5, and 20 μg/ml), Lp2A-I particles with a molar POPC/apoA-I ratio of 40:1 (1, 5, and 20 μg/ml), or Lp2A-I particles with a molar POPC/apoA-I ratio of 80:1 (1, 5, and 20 μg/ml). White bars, 1 μg/ml; gray bars, 5 μg/ml; black bars, 20 μg/ml. (C) eNOS activation in BAECs was assessed during 15-minute incubations with control buffer, HDL (20 μg/ml), Lp2A-I particles with a molar POPC/cholesterol/apoA-I ratio of 80:0:1 (20 μg/ml), or Lp2A-I particles with a molar POPC/cholesterol/apoA-I ratio of 80:5:1 (20 μg/ml). Values (mean ± SEM) are expressed relative to basal activity designated as 100%; n = 4. *P < 0.05 versus basal; †P < 0.05 versus no cholesterol.

Figure 3

Figure 3

Interventions that decrease SR-BI efflux capacity attenuate eNOS stimulation. (A) BAECs were preincubated with control IgG or SR-BI blocking antibody for 30 minutes, and eNOS activation was assessed during 15-minute incubations with control buffer or HDL (50 μg/ml). (B) COS-M6 cells were transfected with eNOS cDNA and either wild-type SR-BI or AVI mutant SR-BI cDNA. Forty-eight hours following transfection, eNOS activation was assessed during 15-minute incubations with control buffer, HDL (20 and 50 μg/ml), or SUVs (250 or 500 μg/ml). Values (mean ± SEM) are expressed relative to basal activity designated as 100%; n = 4. *P < 0.05 vs. basal; †P < 0.05 versus no SR-BI Ab; #P < 0.05 versus wild-type SR-BI.

Figure 4

Figure 4

SR-BI is required for HDL-mediated eNOS activation. (A) COS-M6 cells were transfected with eNOS cDNA and either SR-BI or CD36 cDNA. Forty-eight hours after transfection, eNOS activation was assessed by measuring [3H]

L

-arginine to [3H]

L

-citrulline conversion during 15-minute incubations with control buffer or HDL (50 μg/ml). *P < 0.05 versus basal; †P < 0.05 versus SR-BI. (B) COS-M6 cells were transfected with eNOS cDNA and either sham plasmid or SR-BI cDNA. Forty-eight hours after transfection, eNOS activation was assessed during 15-minute incubations with control buffer, HDL (50 μg/ml), or CD (2%). *P < 0.05 versus basal; #P < 0.05 versus sham. Values (mean ± SEM) are expressed relative to basal activity designated as 100%; n = 4.

Figure 5

Figure 5

SR-BI C-terminal cytoplasmic domain is required for eNOS phosphorylation and activation. COS-M6 cells were transfected with eNOS cDNA and either wild-type SR-BI, SR-BII, or SR-BIΔ509 cDNA. For detection of eNOS phosphorylation (top), 24 hours after transfection the cells were starved in serum-free DMEM for another 24 hours, the cells were incubated with HDL (50 μg/ml) for 0–10 minutes, and cell lysates were analyzed by Western blotting using anti–phospho-eNOS–specific (Ser1179-specific) antibody (peNOS), anti-eNOS monoclonal antibody (eNOS), and anti–SR-BI (extracellular domain) polyclonal antibody (Receptor). For detection of eNOS activation (bottom), parallel sets of transfected cells were used, and [3H]

L

-arginine conversion to [3H]

L

-citrulline was measured during 15-minute incubations with control buffer or HDL (50 μg/ml). Values (mean ± SEM) are expressed as percent of HDL-stimulated activity above basal activity; n = 4. *P < 0.05 versus wild-type SR-BI.

Figure 6

Figure 6

Schematic diagram of SR-BI, CD36, and chimeric receptors. The SR-BI–derived sequence is shown in gray, and the CD36–derived sequence is shown in black.

Figure 7

Figure 7

SR-BI C-terminal cytoplasmic domain is not sufficient for eNOS activation, and the C-terminal transmembrane domain, which binds cholesterol, is also required. (A) COS-M6 cells were transfected with eNOS cDNA and either wild-type SR-BI, CD36, CD/SRCT, or CD/SRCTMT cDNA. For detection of eNOS phosphorylation (top), 24 hours after transfection, the cells were starved in serum-free DMEM for another 24 hours and incubated with HDL (50 μg/ml) for 0 or 10 minutes, and cell lysates were analyzed by Western blotting using anti–phospho-eNOS–specific (Ser1179-specific) antibody and anti-eNOS monoclonal antibody. For detection of eNOS activation (bottom), parallel sets of transfected cells were used, and [3H]

L

-arginine conversion to [3H]

L

-citrulline was measured during 15-minute incubations with control buffer or HDL (50 μg/ml). Values (mean ± SEM) are expressed as percent of HDL-stimulated activity above basal activity; n = 4. *P < 0.05 versus wild-type SR-BI. (B) HEK 293 cells were transfected and labeled with [3H]photocholesterol; receptors were immunoprecipitated with antibody to the SR-BI C-terminal tail; and the [3H]photocholesterol associated with the receptor was assessed by autoradiography (top). The abundance of immunoprecipitated receptor was assessed by Western blotting (bottom), and the relative amount of [3H]photocholesterol-bound receptor versus total receptor was calculated.

Figure 8

Figure 8

SR-BI binds either plasma membrane or lysosomal membrane cholesterol. HEK 293 cells were transfected with SR-BI, CD/SRCT, LIMP-II/SRCT, SR-BI/LIICT, CD36/LIICT, or LIMP-II and labeled with [3H]photocholesterol; plasma membranes or lysosomal membranes were isolated; receptors were immunoprecipitated with antibody to the SR-BI C-terminal tail or to LIMP-II; and the [3H]photocholesterol associated with the receptor was assessed by autoradiography (top). The abundance of immunoprecipitated receptor was assessed by Western blotting (bottom), and the relative amount of [3H]photocholesterol-bound receptor versus total receptor was calculated.

Figure 9

Figure 9

CD36 extracellular domain is capable of transducing signal if SR-BI transmembrane and cytoplasmic domains are present. (A) COS-M6 cells were transfected with eNOS cDNA and either wild-type SR-BI, CD/SRCT, SR/CDECL/SR, or SR/CDTM/SR cDNA. For detection of eNOS phosphorylation (left), 24 hours after transfection the cells were starved in serum-free DMEM for another 24 hours, the cells were incubated with HDL (50 μg/ml) for 0 or 10 minutes, and cell lysates were analyzed by Western blotting using anti–phospho-eNOS–specific (Ser1179-specific) antibody and anti-eNOS monoclonal antibody. For detection of eNOS activation (right), parallel sets of transfected cells were used, and [3H]

L

-arginine conversion to [3H]

L

-citrulline was measured during 15-minute incubations with control buffer or HDL (50 μg/ml). Values (mean ± SEM) are expressed as percent of HDL-stimulated activity above basal activity; n = 4. *P < 0.05 versus wild-type SR-BI. (B and C) HEK 293 cells were transfected with the constructs shown in A and labeled with [3H]photocholesterol; receptors were immunoprecipitated with antibody to the SR-BI C-terminal tail; and the [3H]photocholesterol associated with the receptor was assessed by autoradiography (top). The abundance of immunoprecipitated receptor was assessed by Western blotting (bottom), and the relative amount of [3H]photocholesterol-bound receptor versus total receptor was calculated.

References

    1. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu. Rev. Biochem. 1983;52:223–261. - PubMed
    1. Gordon DJ, Rifkind BM. High-density lipoprotein — the clinical implications of recent studies. N. Engl. J. Med. 1989;321:1311–1316. - PubMed
    1. Stein O, Stein Y. Atheroprotective mechanisms of HDL. Atherosclerosis. 1999;144:285–301. - PubMed
    1. Trigatti B, Rigotti A, Krieger M. The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism. Curr. Opin. Lipidol. 2000;11:123–131. - PubMed
    1. Calvo D, Vega MA. Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J. Biol. Chem. 1993;268:18929–18935. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources