Massimo Sargiacomo

Interaction of a Receptor Tyrosine Kinase, EGF-R, with Caveolins CAVEOLIN BINDING NEGATIVELY REGULATES TYROSINE AND SERINE/THREONINE KINASE ACTIVITIES

Caveolin, a 21–24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein α subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinasein vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.

Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis

Caveolae, 50-100 nm invaginations that represent a subcompartment of the plasma membrane, have been known for many years, but their exact roles remain uncertain. The findings that the caveolae coat protein caveolin is a v-Src substrate and that G-protein-coupled receptors are present in caveolae have suggested a relationship between caveolae, caveolin and transmembrane signalling. The recent isolation of caveolin-rich membrane domains in which caveolin exists as a hetero-oligomeric complex with integral membrane proteins and known cytoplasmic signalling molecules provides support for this hypothesis. Compartmentalization of certain signalling molecules within caveolae could allow efficient and rapid coupling of activated receptors to more than one effector system.

Microenvironmental pH is a key factor for exosome traffic in tumor cells.

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models.

Organization of plasma membrane functional rafts upon T cell activation.

Raft microdomains have been shown to play a key role in T cell activation. We found that in human T lymphocytes the formation of functional rafts at the plasma membrane was induced by T cell priming. In resting T cells from peripheral blood Lck and the raft glycosphingolipid GM1 resided in intracellular membranes. T cell activation induced synthesis of GM1 and effector cells showed very high levels of this lipid, which became predominantly plasma membrane associated. TCR triggering also induced targeting of the cytosolic Lck to the plasma membrane. Thus, effector cells acquire an improved signaling machinery by increasing the amount of rafts at the plasma membrane. The fact that, when compared with naive T cells, memory T cells showed higher GM1 levels suggests that raft lipid synthesis may be developmentally regulated and tune T cell responsiveness.

TfR2 localizes in lipid raft domains and is released in exosomes to activate signal transduction along the MAPK pathway.

Transferrin receptor 2 (TfR2) possesses a YQRV motif similar to the YTRF motif of transferrin receptor 1 (TfR1) responsible for the internalization and secretion through the endosomal pathway. Raft biochemical dissection showed that TfR2 is a component of the low-density Triton-insoluble (LDTI) plasma membrane domain, able to co-immunoprecipitate with caveolin-1 and CD81, two structural raft proteins. In addition, subcellular fractionation experiments showed that TfR1, which spontaneously undergoes endocytosis and recycling, largely distributed to intracellular organelles, whereas TfR2 was mainly associated with the plasma membrane. Given the TfR2 localization in lipid rafts, we tested its capability to activate cell signalling. Interaction with an anti-TfR2 antibody or with human or bovine holotransferrin showed that it activated ERK1/ERK2 and p38 MAP kinases. Integrity of lipid rafts was required for MAPK activation. Co-localization of TfR2 with CD81, a raft tetraspanin exported through exosomes, prompted us to investigate exosomes released by HepG2 and K562 cells into culture medium. TfR2, CD81 and to a lesser extent caveolin-1, were found to be part of the exosomal budding vesicles. In conclusion, the present study indicates that TfR2 localizes in LDTI microdomains, where it promotes cell signalling, and is exported out of the cells through the exosome pathway, where it acts as an intercellular messenger.

Role of Cholesterol in Human Immunodeficiency Virus Type 1 Envelope Protein-Mediated Fusion with Host Cells

ABSTRACT In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-β-cyclodextrin (MβCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1α- and macrophage inflammatory protein 1β-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MβCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.

Mutational analysis of caveolin-induced vesicle formation Expression of caveolin-1 recruits caveolin-2 to caveolae membranes

Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50–100 nm. Although recombinant expression of caveolin‐1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin‐caveolin interactions regulate the size of caveolae, as caveolin‐1 undergoes two stages of self‐oligomerization. To test this hypothesis directly, we have created two caveolin‐1 deletion mutants that lack regions of caveolin‐1 that are involved in directing the self‐assembly of caveolin‐1 oligomers. More specifically, Cav‐1 Δ61–100 lacks a region of the N‐terminal domain that directs the formation of high molecular mass caveolin‐1 homo‐oligomers, while Cav‐1 ΔC lacks a complete C‐terminal domain that is required to allow caveolin homo‐oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav‐1 Δ61–100 and Cav‐1 ΔC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500–1000 nm. In addition, caveolin‐induced vesicle formation appears to be isoform‐specific. Recombinant expression of caveolin‐2 under the same conditions failed to drive the formation of vesicles, while caveolin‐3 expression yielded caveolae‐sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin‐1 expression, but continue to express caveolin‐2, no morphologically distinguishable caveolae are observed. In addition, as caveolin‐2 alone exists mainly as a monomer or homo‐dimer, while caveolins 1 and 3 exist as high molecular mass homo‐oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae‐sized vesicles. In direct support of this notion, regulated induction of caveolin‐1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin‐2 to caveolae membranes. The ability of caveolin‐1 to recruit caveolin‐2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co‐expressed and interact with each other to form high molecular mass hetero‐oligomers containing both caveolins 1 and 2.

Caveolae purification and glycosylphosphatidylinositol-linked protein sorting in polarized epithelia.

Publisher Summary This chapter describes the techniques used for the recombinant expression of glycosylphosphatidylinositol (GPI)-linked proteins in epithelial cell lines and the measurement of cell-surface polarity of endogenous or transfected GPI-linked proteins at steady state and during transport. The chapter also discusses the methods for purification and characterization of caveolae from cultured cells. To study the sorting of endogenous GPI-linked proteins in polarized cells, a series of cell-surface labeling techniques that allow the rapid biochemical determination of the polarity of a given cell-surface antigen is developed. Such labeling techniques depend on the growth of polarized cells on permeable supports that allow for separate access to the apical and basolateral domains. These techniques are then applied to a variety of available intestinal and renal epithelial cell lines, such as the Madin-Darby canine kidney (MDCK), LLC-PK1, Caco-2, and SK-C015 lines, that spontaneously form polarized monolayers in culture. The GPI-linked proteins are detected by their sensitivity to release by treatment with bacterial PI-specific phospholipase C. To measure the polarized sorting of the recombinant proteins during cell-surface transport, additional assays are developed to monitor the cell surface delivery, endocytosis, and transcytosis.

Porin Is Present in the Plasma Membrane Where It Is Concentrated in Caveolae and Caveolae-related Domains

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.