Amnon Schlegel

Caveolins, a Family of Scaffolding Proteins for Organizing “Preassembled Signaling Complexes” at the Plasma Membrane

Caveolae are vesicular invaginations of the plasma membrane. The chief structural proteins of caveolae are the caveolins. Caveolins form a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules. Because the responsibilities assigned to caveolae continue to increase, this review will focus on: (i) caveolin structure/function and (ii) caveolae-associated signal transduction. Studies that link caveolae to human diseases will also be considered.

Caveolae, Plasma Membrane Microdomains for α-Secretase-mediated Processing of the Amyloid Precursor Protein

Caveolae are plasma membrane invaginations where key signaling elements are concentrated. In this report, both biochemical and histochemical analyses demonstrate that the amyloid precursor protein (APP), a source of Aβ amyloid peptide, is enriched within caveolae. Caveolin-1, a principal component of caveolae, is physically associated with APP, and the cytoplasmic domain of APP directly participates in this binding. The characteristic C-terminal fragment that results from APP processing by α-secretase, an as yet unidentified enzyme that cleaves APP within the Aβ amyloid sequence, was also localized within these caveolae-enriched fractions. Further analysis by cell surface biotinylation revealed that this cleavage event occurs at the cell surface. Importantly, α-secretase processing was significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current results indicate that caveolae and caveolins may play a pivotal role in the α-secretase-mediated proteolysis of APP in vivo.

A Role for the Caveolin Scaffolding Domain in Mediating the Membrane Attachment of Caveolin-1 THE CAVEOLIN SCAFFOLDING DOMAIN IS BOTH NECESSARY AND SUFFICIENT FOR MEMBRANE BINDING IN VITRO

Here, we have created a series of caveolin-1 (Cav-1) deletion mutants to examine whether the membrane spanning segment is required for membrane attachment of caveolin-1 in vivo. One mutant, Cav-1-(1–101), contains only the cytoplasmic N-terminal domain and lacks the membrane spanning domain and the C-terminal domain. Interestingly, Cav-1-(1–101) still behaves as an integral membrane protein but lacks any known signals for lipid modification. In striking contrast, another deletion mutant, Cav-1-(1–81), behaved as a soluble protein. These results implicate caveolin-1 residues 82–101 (also known as the caveolin scaffolding domain) in membrane attachment. In accordance with the postulated role of the caveolin-1 scaffolding domain as an inhibitor of signal transduction, Cav-1-(1–101) retained the ability to functionally inhibit signaling along the p42/44 mitogen-activated protein kinase cascade, whereas Cav-1-(1–81) was completely ineffective. To rule out the possibility that membrane attachment mediated by the caveolin scaffolding domain was indirect, we reconstituted the membrane binding of caveolin-1 in vitro. By using purified glutathioneS-transferase-caveolin-1 fusion proteins and reconstituted lipid vesicles, we show that the caveolin-1 scaffolding domain and the C-terminal domain (residues 135–178) are both sufficient for membrane attachment in vitro. However, the putative membrane spanning domain (residues 102–134) did not show any physical association with membranes in this in vitro system. Taken together, our results provide strong evidence that the caveolin scaffolding domain contributes to the membrane attachment of caveolin-1.

Caveolin-1 potentiates estrogen receptor α (ERα) signaling. Caveolin-1 drives ligand-independent nuclear translocation and activation of ERα

Estrogen receptor α (ERα) is a soluble protein that mediates the effects of the gonadal estrogens such as 17β-estradiol. Upon ligand binding, a cytoplasmic pool of ERα translocates to the nucleus, where it acts as a transcription factor, driving the expression of genes that contain estrogen-response elements. The activity of ERα is regulated by a number of proteins, including cytosolic chaperones and nuclear cofactors. Here, we show that caveolin-1 potentiates ERα-mediated signal transduction. Coexpression of caveolin-1 and ERα resulted in ligand-independent translocation of ERα to the nucleus as shown by both cell fractionation and immunofluorescence microscopic studies. Similarly, caveolin-1 augmented both ligand-independent and ligand-dependent ERα signaling as measured using a estrogen-response element-based luciferase reporter assay. Caveolin-1-mediated activation of ERα was sensitive to a well known ER antagonist, 4-hydroxytamoxifen. However, much higher concentrations of tamoxifen were required to mediate inhibition in the presence of caveolin-1. Interestingly, caveolin-1 expression also synergized with a constitutively active, ligand-independent ERα mutant, dramatically illustrating the potent stimulatory effect of caveolin-1 in this receptor system. Taken together, our results identify caveolin-1 as a new positive regulator of ERα signal transduction.

REMOVED: The caveolin triad: caveolae biogenesis, cholesterol trafficking, and signal transduction

Caveolins are a family of proteins that coat the cytoplasmic face of caveolae, vesicular invaginations of the plasma membrane. These proteins are central to the organization of the proteins and lipids that reside in caveolae. Caveolins transport cholesterol to and from caveolae, and they regulate the activity of signaling proteins that reside in caveolae. Through studying the genes encoding the caveolae coat proteins, we have learned much about how they perform these multiple functions.

Ligand-independent activation of oestrogen receptor alpha by caveolin-1.

Expression of caveolin-1 in the human mammary adenocarcinoma cell line MCF-7 causes ligand-independent concentration of oestrogen receptor alpha (ERalpha) in the nucleus, and potentiates ligand-independent and ligand-dependent transcription from an oestrogen response element-driven reporter gene. Furthermore, caveolin-1 co-immunoprecipitates with ERalpha [Schlegel, Wang, Katzenellenbogen, Pestell and Lisanti (1999) J. Biol. Chem. 274, 33551-33556]. In the present study we show that caveolin-1 binds directly to ERalpha. This interaction is mediated by residues 82-101 of caveolin-1 (i.e. the caveolin scaffolding domain) and residues 1-282 of ERalpha. The caveolin-binding domain of ERalpha includes the ligand-independent transactivation domain, activation function (AF)-1, but lacks the hormone-binding domain and the ligand-gated transactivation domain, AF-2. In co-transfection studies, caveolin-1 potentiates the transcriptional activation of ERalpha(1-282), a truncation mutant that has intact AF-1 and DNA-binding domains. Since AF-1 activity is regulated largely by phosphorylation we determined that co-expression with caveolin-1 increased the basal phosphorylation of ERalpha(1-282), but blocked the epidermal growth factor-dependent increase in phosphorylation. Indeed, caveolin-1 interacted with and potentiated the transactivation of an ERalpha mutant that cannot be phosphorylated by extracellular signal-regulated kinase (ERK)1/2 [ERalpha(Ser(118)-->Ala)]. Thus caveolin-1 is a novel ERalpha regulator that drives ERK1/2-independent phosphorylation and activation of AF-1.

Withdrawal of ?Caveolae and Their Coat Proteins, the Caveolins: From Electron Microscopic Novelty to Biological Launching Pad? [Journal of Cellular Physiology 186(3) 329-337 (2001)]

The Publisher regrets that this review article must be withdrawn owing to an issue of multiple publication. The manuscript was submitted in nearly identical form to three journals (Frontiers in Bioscience, Cytokine and Growth Factor Reviews and Journal of Cellular Physiology) and was accepted and published by all three. As the article was accepted and published in Frontiers in Bioscience first, the copyright belongs to that journal. The bibliographic details of the original review article published in Frontiers in Bioscience are: “Caveolins in Cholesterol Trafficking and Signal Transduction: Implications for Human Disease” Amnon Schlegel et al. Frontiers in Bioscience 2000 Dec 1; Volume 5:D929‐37 http://www.bioscience.org/2000/v5/d/schlegel/list.htm

Withdrawal of “The caveolin triad: caveolae biogenesis, cholesterol trafficking, and signal transduction” [Cytokine & Growth Factor Reviews 12(1) (2001) 41–51]

The Publisher regrets that this review article must be withdrawn owing to an issue of multiple publication. The manuscript was submitted in nearly identical form to three journals (Frontiers in Bioscience, Journal of Cellular Physiology and Cytokine & Growth Factor Reviews) and was accepted and published by all three. As the article was accepted and published in Frontiers in Bioscience first, the copyright belongs to that journal. The bibliographic details of the original review article published in Frontiers in Bioscience are: