Cholesterol Depletion Inhibits Epidermal Growth Factor Receptor Transactivation by Angiotensin II in Vascular Smooth Muscle Cells

111

The mechanisms involved in angiotensin II type 1 receptor (AT 1 -R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT 1 -R is not concentrated in caveolae but is clustered in cholesterolindependent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT 1 -R in caveolae, AT 1 -R⅐caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT 1 -R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT 1 -R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT 1 -R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT 1 -R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT 1 -R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT 1 -R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT 1 -R through the exocytic pathway, but this does not result in AT 1 -R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT 1 -R.Lipid-based sorting mechanisms play an important role in the organization of the plasma membrane into microdomains (1-3). The biophysical properties of sphingolipids and cholesterol drive the spontaneous formation of lateral assemblies of liquid-ordered lipid rafts in a sea of liquid-disordered phospholipids. The biological importance of lipid rafts follows from the lateral segregation that they impose on membrane proteins. The differential distribution of plasma membrane proteins across raft and nonraft membranes in turn results in the concentration of specific groups of signaling proteins and lipids within discrete areas of the cell membrane (3-6). This increases the efficiency and specificity of signaling events by allowing more efficient interactions between proteins and by preventing cross-talk between different pathways.Caveolae are an abundant surface feature of many mammalian cells and represent a specific subtype of lipid raft. Functionally, caveolae have been implicated in endocytosis (7), potocytosis (8), transcytosis (9), apical transport (10), and cholesterol balance (11). Caveolae are identified by their characteristic morphology (flask-shaped, 55-65-nm diameter pits) and the presence of integral membrane proteins, termed caveolins, of which three mammalian isoforms have bee...

Section: Figsupporting

confidence: 48%

Caveolin Interacts with the Angiotensin II Type 1 Receptor during Exocytic Transport but Not at the Plasma Membrane

Wyse

Prior

Qian

et al. 2003

111

“…1B, p Ͼ 0.05), suggesting that the glucose effect on signal transduction might be specific to AngII. As shown previously (14,15), Akt and ERK1/2 phosphorylation upon stimulation with AngII mostly depended on EGFR transactivation, because AG1478 (1 M), a tyrosine kinase inhibitor specific for EGFR, reduced AngII-induced Akt and ERK1/2 phosphorylation by 111.6% and 78.9%, respectively, in HGcultured cells (data not shown).…”

Section: Glucose Modulates Akt and Erk1/2 Phosphorylation Uponmentioning

confidence: 77%

“…Membrane-A previous report demonstrated that disruption of lipid rafts and/or caveolae prevented EGFR transactivation by AngII but not activation by EGF (15). It was also shown that the second cysteinerich region of EGFR containing two potential N-glycosylation sites was important for targeting EGFR to lipid rafts and/or caveolae (28).…”

Section: Localization Of Egfr Within Plasmamentioning

confidence: 99%

Epidermal Growth Factor Receptor Transactivation Is Regulated by Glucose in Vascular Smooth Muscle Cells

Konishi

Berk

2003

We hypothesized that glucose-mediated alterations in vascular smooth muscle cell signal transduction contribute to diabetic complications. We found enhanced AngII activation of Akt and extracellular ERK1/2 in vascular smooth muscle cells incubated with high glucose (27.5 mM) compared with low glucose (5.5 mM). Because AngII-mediated transactivation of the epidermal growth factor receptor (EGFR) is important in Akt and ERK1/2 activation, we studied the effects of glucose on EGFR function. The EGFR in cells cultured for 48 h in low glucose was smaller (145 kDa) than the EGFR in cells cultured with high glucose (170 kDa). The shift from the 170-kDa isoform to the 145-kDa isoform was reversible and dependent upon glucose concentration with EC 50 ϳ 1 mM. N-Glycosylation was responsible because peptide N-glycosidase F treatment of isolated 170-kDa EGFR yielded a single band at 145 kDa. Cell surface biotinylation showed that the 145-kDa EGFR was present on plasma membrane. AngII and other G-protein-coupled receptor ligands known to transactivate EGFR phosphorylated the 170-kDa EGFR but not the 145-kDa EGFR, whereas EGF, heparin-binding EGF-like growth factor, and transforming growth factor-␣ phosphorylated both receptors. Subcellular fractionation showed that the 145-kDa receptor localized to a different plasma membrane domain than the 170-kDa receptor. These results establish a novel mechanism by which glucosedependent EGFR N-glycosylation modulates AngII signal transduction and suggest a potential mechanism for pathogenic effects of AngII in diabetic vasculopathy.

“…Cav-1 is phosphorylated on Tyr-14 by Src, Fyn, and Abl in response to insulin, angiotensin II, osmotic shock, oxidative stress, and IGF-I (44,45,(53)(54)(55)(56). In MM cells, IL-6 stimulates Src and p59Fyn, p56/59Hck, and p56Lyn activation (57).…”

Section: Cav-1 Tyrosine Phosphorylation Induced By Il-6 and Igf-i Is mentioning

confidence: 99%

Essential Role of Caveolae in Interleukin-6- and Insulin-like Growth Factor I-triggered Akt-1-mediated Survival of Multiple Myeloma Cells

Podar

Tai

Cole

et al. 2003

133

103

Caveolae, specialized flask-shaped lipid rafts on the cell surface, are composed of cholesterol, sphingolipids, and structural proteins termed caveolins; functionally, these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. In the present study, we examined the role of caveolin-1 in multiple myeloma cells. We show for the first time that caveolin-1, which is usually absent in blood cells, is expressed in multiple myeloma cells. Analysis of myeloma cell-derived plasma membrane fractions shows that caveolin-1 is co-localized with interleukin-6 receptor signal transducing chain gp130 and with insulin-like growth factor-I receptor. Cholesterol depletion by ␤-cyclodextrin results in the loss of caveola structure in myeloma cells, as shown by transmission electron microscopy, and loss of caveolin-1 function. Interleukin-6 and insulin-like growth factor-I, growth and survival factors in multiple myeloma, induce caveolin-1 phosphorylation, which is abrogated by pre-treatment with ␤-cyclodextrin. Importantly, inhibition of caveolin-1 phosphorylation blocks both interleukin-6-induced protein complex formation with caveolin-1 and downstream activation of the phosphatidylinositol 3-kinase/ Akt-1 pathway. ␤-Cyclodextrin also blocks insulin-like growth factor-I-induced tyrosine phosphorylation of insulin-responsive substrate-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. Therefore, cholesterol depletion by ␤-cyclodextrin abrogates both interleukin-6-and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1. Taken together, this study identifies caveolin-1 and other structural membrane components as potential new therapeutic targets in multiple myeloma.