Richard G. W. Anderson

Caveolin, a protein component of caveolae membrane coats

Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.

Lipid rafts: At a crossroad between cell biology and physics

Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes and the notion of lipid rafts gives specificity to this broad concept. However, the lipid raft field is now at a technical impasse because the physical tools to study biological membranes as a liquid that is ordered in space and time are still being developed. This has lead to a disconnection between the concept of lipid rafts as derived from biochemical and biophysical assays and their existence in the cell. Here, we compare the concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes. Further application of existing tools and the development of new tools are needed to understand the dynamic heterogeneity of biological membranes.

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide–dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

A Role for Caveolin in Transport of Cholesterol from Endoplasmic Reticulum to Plasma Membrane

Caveolin is a 22-kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half-time of ∼10 min. The cholesterol then rapidly flowed from caveolae to non-caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 μg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting cholesterol transport is linked to caveolin movement. Caveolae fractions from cells expressing caveolin were enriched in cholesterol 3-4-fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.

Multiple Functions of Caveolin-1

The amino acid sequence of caveolin-1 predicts that it is an integral membrane protein, and there is strong experimental evidence that it has this property. For example, caveolin-1 is cotranslationally inserted into the ER and shipped to the Golgi apparatus where it is incorporated into lipid domains that sort molecules for shipment to the cell surface (1). The preferred location for caveolin-1 at the cell surface is the caveola, and it cannot be removed from these membranes without detergent (2). Finally, the movements of green fluorescent protein-tagged caveolin-1 suggest that normally caveolin-1 moves with caveolae-derived vesicles to multiple interior compartments and then recycles back to the cell surface (3). By contrast, there is compelling evidence that caveolin-1 can be a soluble protein. Immunogold labeling first detected soluble caveolin-1 in the lumen of the ER after cells were exposed to cholesterol oxidase (4). Then a small pool of soluble caveolin-1 was found in fibroblast cytosol in a complex with chaperones (5). A routine survey of caveolin-1 distribution in different tissues identified cells that targeted caveolin-1 primarily to the cytosol (skeletal muscle cells and keratinocytes), to the lumen of secretory vesicles (serous cells of pancreas, fundic stomach, and salivary gland), and to mitochondria (airway epithelial cells and hepatocytes) (6, 7). Both the secreted and the cytosolic caveolin-1 appear to be embedded in lipoprotein-like particles, which may explain why they are soluble. Thus, caveolin-1 is an unusual protein that can be both an integral membrane protein and soluble in multiple cellular compartments. We believe this property is an important clue about its function.

Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling.

In nerve terminals, Ca(2+)-stimulated synaptic vesicle exocytosis is rapidly followed by endocytosis. Synaptic vesicle endocytosis requires clathrin-coated pits similar to receptor-mediated endocytosis in fibroblasts. Binding of clathrin AP-2 (adaptor complex) to an unidentified high affinity membrane receptor appears to be necessary for coated pit assembly in fibroblasts. We now show that synaptic vesicles have a high affinity AP-2 site (KD, approximately 1 x 10(-10) M) similar to the one observed in fibroblasts. Using a combination of competition and direct binding assays, we demonstrate that synaptotagmin I, an intrinsic membrane protein of synaptic vesicles, has all of the properties of the AP-2 receptor and that AP-2 binds to the second C2 domain in the molecule. Thus, synaptotagmin I may be a multifunctional protein with a function in endocytosis in addition to the previously proposed role in exocytosis.

Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates: implications for the Cori cycle.

Lactate and pyruvate cross cell membranes via a monocarboxylate transporter (MCT) with well-defined properties but undefined molecular structure. We report the cloning of a cDNA encoding MCT1, a monocarboxylate transporter whose properties resemble those of the erythrocyte MCT, including proton symport, trans acceleration, and sensitivity to alpha-cyanocinnammates. A Phe to Cys substitution in MCT1 converts it to Mev, a mevalonate transporter. MCT1 is abundant in erythrocytes, cardiac muscle, and basolateral intestinal epithelium. In skeletal muscle it is restricted to mitochondria-rich myocytes. As sperm traverse the epididymis, MCT1 switches from sperm to epithelial cells. MCT1 is present at low levels in liver, suggesting another MCT in this tissue. By exporting lactate from intestine and erythrocytes, MCT1 participates in the Cori cycle. It also participates in novel pathways of monocarboxylate metabolism in muscle and sperm.

Localization of Epidermal Growth Factor-stimulated Ras/Raf-1 Interaction to Caveolae Membrane

An essential step in the epidermal growth factor (EGF)-dependent activation of MAP kinase is the recruitment of Raf-1 to the plasma membrane. Here we present evidence that caveolae are the membrane site where Raf-1 is recruited. Caveolae fractions prepared from normal Rat-1 cells grown in the absence of serum were highly enriched in both EGF receptors and Ras. Thirty seconds after EGF was added to these cells Raf-1 began to appear in caveolae but not in non-caveolae membrane fractions. The maximum concentration was reached at 3 min followed by a decline over the next 60 min. During this time EGF receptors disappeared from the caveolae fraction while the concentration of Ras remained constant. The Raf-1 in this fraction was able to phosphorylate MAP kinase kinase, whereas cytoplasmic Raf-1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf-1 to caveolae. Overexpression of Ha-Ras caused the recruitment of Raf-1 to caveolae independently of EGF stimulation, and this was blocked by the farnesyltransferase inhibitor BZA-5B. Finally, prenylation appeared to be required for localization of Ras to caveolae.

Role of plasmalemmal caveolae in signal transduction

Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules. They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 degreesC. Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules. This includes G protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules. More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface. This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules. They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4°C. Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules. This includes G protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules. More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface. This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.

Cholesterol Depletion of Caveolae Causes Hyperactivation of Extracellular Signal-related Kinase (ERK)

Previously we showed that activation of Erk in quiescent cells occurs in the caveolae fraction isolated from fibroblasts. Since the structure and function of caveolae is sensitive to the amount of cholesterol in the membrane, it might be that a direct link exists between the concentration of membrane cholesterol and mitogen-activated protein (MAP) kinase activation. We acutely lowered the cholesterol level of the caveolae fraction by incubating Rat-1 cells in the presence of either cyclodextrin or progesterone. Cholesterol-depleted caveolae had a reduced amount of several key protein components of the MAP kinase complex, including Ras, Grb2, Erk2, and Src. Incubation of these cells in the presence of epidermal growth factor (EGF) caused a rapid loss of EGF receptor from the caveolae fraction, but the usual recruitment of c-Raf was markedly inhibited. Despite the reduced amount of c-Raf and Erk2 in the cholesterol-depleted caveolae fraction, EGF caused a hyperactivation of the remaining caveolae Erk isoenzymes. This was followed by an increase in the amount of active Erk in the cytoplasm. The increased amount of activated Erk produced under these conditions was linked to a 2-fold higher level of EGF-stimulated DNA synthesis. Even cholesterol depletion by itself stimulated Erk activation and DNA synthesis. These results suggest that the MAP kinase pathway can connect the cholesterol level of caveolae membrane to the control of cell division.