Co-purification and Direct Interaction of Ras with Caveolin, an Integral Membrane Protein of Caveolae Microdomains

Song, Kenneth S.; Li, Shengwen; Okamoto, Takashi; Quilliam, Lawrence A.; Sargiacomo, Massimo; Lisanti, Michael P.

doi:10.1074/jbc.271.16.9690

Cited by 969 publications

(1,012 citation statements)

References 56 publications

(121 reference statements)

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“…Sucrose density membrane fractionation under detergent-free conditions A detergent-free method was used to isolate caveolaeenriched domains and prepare subcellular fractions as previously described [34] but with slight modifications. Treated and control cells from T-75 cm 2 flasks were washed in icecold PBS (pH 7.3) and the cells scraped into 1 ml of 150 mM Na 2 CO 3 with 1 mM EDTA (pH 11.0).…”

Section: Rt-pcrmentioning

confidence: 99%

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Thomas

Hutcheson

Campbell

et al. 2009

Breast Cancer Res Treat

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Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance. Breast Cancer Research and Treatment, Springer Verlag, 2009, 119 (3) Abstract Caveolin-1 displays both tumour-suppressor and tumour-promoter properties in breast cancer. Using characterised preclinical cell models for the transition of oestrogen-sensitive (WT-MCF-7 cells) to a tamoxifenresistant (TAM-R cells) phenotype we examined the role caveolin-1 in the development of hormone-resistant breast cancer. The WT-MCF-7 cells showed abundant expression of caveolin-1 which potentiated oestrogen-receptor (ERa) signalling and promoted cell growth despite caveolin-1 mediating inhibition of ERK signalling. In TAM-R cells caveolin-1 expression was negligible, repressed by EGF-R/ERK signalling. Pharmacological inhibition of EGFR/ERK in TAM-R cells restored caveolin-1 and also resulted in the emergence of pools of phosphorylated caveolin-1. WT-MCF-7 cells exposed to tamoxifen for upto 12 weeks displayed increased caveolin-1 (peaking by week 2) followed (after week 8) by a marked decrease as the cells progress to develop a stable tamoxifen-resistant phenotype. The targeted down-regulation (siRNA) of caveolin-1 in WT-MCF-7 cells reduced growth but did not affect their sensitivity to tamoxifen, suggesting loss of caveolin-1 alone is not sufficient to confer tamoxifen-resistance. Hyperactivation of EGFR/ERK is a feature of tamoxifen-resistant breast cancer cells, a principal driver of cell growth. Recombinant expression of caveolin-1 in TAM-R cells did not affect EGFR/ERK activity, potentially due to mislocalisation of caveolin-1 through hyperactivation of the mTOR pathway or altered caveolin-1 phosphorylation. This work defines a novel role for caveolin-1 with implications for the clinical course of breast cancer and identifies caveolin-1 as a potential drug target for the treatment of early oestrogen-dependent breast cancers. Further, the loss of caveolin-1 may have benefit as a molecular signature for tamoxifen resistance.

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Section: Rt-pcrmentioning

confidence: 99%

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Thomas

Hutcheson

Campbell

et al. 2009

Breast Cancer Res Treat

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“…A modified version of our published procedure was used to prepare lipid rafts (Xu et al, 2006;Huang et al, 2007), which was originally described by Song et al (1996). Prepared by this method, non-caveolae rafts cannot be distinguished from caveolae.…”

Section: Mor In Rat Brain Cpu and Rmor Expressed In Cho Cells Are Maimentioning

confidence: 99%

Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells

Huang

Yoon

et al. 2007

Brain Research

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Lipid rafts are small cholesterol-and glycosphingolipid-enriched membrane subdomains. Here we compared the mu opioid receptor (MOR)-lipid rafts relationship in the rat brain, where neurons have non-caveolae rafts, and in CHO cells stably transfected with HA-rat MOR (CHO-HArMOR), which are enriched in caveolae. Membranes of rat caudate putamen (CPu) and thalamus or CHO-HA-rMOR cells were homogenized, sonicated in a detergent-free 0.5 M Na 2 CO 3 buffer and fractionated through sucrose density gradients. Western blot and [ 3 H]diprenorphine binding showed that ~70% of MOR in CHO-HA-rMOR was present in low-density (5-20% sucrose) fractions enriched in cholesterol and/or ganglioside M1 (GM1) (lipid rafts) in plasma membranes, whereas about 70% and 45% of MOR in CPu and thalamus, respectively, were associated with lipid rafts. Incubation with a saturating concentration of etorphine or morphine at 37 °C for 30 min failed to change the MOR location in rafts in CHO-HA-rMOR, indicating that the internalized MOR does not move out of rafts, in contrast to the delta opioid receptor. In vivo, rafts association of MOR in CPu and thalamus was not affected significantly in rats implanted with two 75-mg morphine pellets for 72 h. In addition, cholesterol reduction by methyl-β-cyclodextrin (MCD) disrupted rafts and shifted MOR to higher density fractions in both CHO-HA-rMOR and CPu membranes. However, MCD treatment had opposite impacts on MOR signaling in the two tissues: it attenuated MOR-mediated [ 35 S]GTPγS binding in CPu but enhanced it in CHO-HA-rMOR.

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“…1999), H‐Ras (Song et al. 1996), G proteins and G‐protein‐coupled receptors, and nitric oxide synthase (Feron et al. 1996) are found in caveolae.…”

Section: Introductionmentioning

confidence: 99%

Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance

et al. 2016

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Insulin resistance and diabetes are comorbidities of obesity and affect one in 10 adults in the United States. Despite the high prevalence, the mechanisms of cardiac insulin resistance in obesity are still unclear. We test the hypothesis that the insulin receptor localizes to caveolae and is regulated through binding to caveolin‐3 (CAV3). We further test whether haploinsufficiency for CAV3 increases the susceptibility to high‐fat‐induced insulin resistance. We used in vivo and in vitro studies to determine the effect of palmitate exposure on global insulin resistance, contractile performance of the heart in vivo, glucose uptake in the heart, and on cellular signaling downstream of the IR. We show that haploinsufficiency for CAV3 increases susceptibility to palmitate‐induced global insulin resistance and causes cardiomyopathy. On the basis of fluorescence energy transfer (FRET) experiments, we show that CAV3 and IR directly interact in cardiomyocytes. Palmitate impairs insulin signaling by a decrease in insulin‐stimulated phosphorylation of Akt that corresponds to an 87% decrease in insulin‐stimulated glucose uptake in HL‐1 cardiomyocytes. Despite loss of Akt phosphorylation and lower glucose uptake, palmitate increased insulin‐independent serine phosphorylation of IRS‐1 by 35%. In addition, we found lipid induced downregulation of CD36, the fatty acid transporter associated with caveolae. This may explain the problem the diabetic heart is facing with the simultaneous impairment of glucose uptake and lipid transport. Thus, these findings suggest that loss of CAV3 interferes with downstream insulin signaling and lipid uptake, implicating CAV3 as a regulator of the IR and regulator of lipid uptake in the heart.

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Co-purification and Direct Interaction of Ras with Caveolin, an Integral Membrane Protein of Caveolae Microdomains

Cited by 969 publications

References 56 publications

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells

Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance

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