Large metal ions (>0.9 A ionic radius) have previously been found to bind only weakly to human serum transferrin (hTF, 80 kDa), presumably because the interdomain cleft cannot close around the metal and synergistic anion. Surprisingly, therefore, we report that Bi3+ (ionic radius 1.03 A), a metal ion widely used in anti-ulcer drugs, binds strongly to both the N- and C-lobes with log K1* = 19.42 and log K2* = 18.58 (10 mM Hepes, 5 mM bicarbonate, 310 K). The uptake of Bi3+ by apo-hTF from bismuth citrate complexes is very slow (hours), whereas that from bismuth nitrilotriacetate is rapid (minutes). Evidence from absorption and NMR spectroscopy is presented to show that Bi3+ binds to the specific Fe3+ binding sites along with carbonate as the synergistic anion. Under the conditions used, preferential binding of Bi3+ to the C-lobe of hTF is observed. Linear free energy relationships show that there is a strong correlation between the strength of binding of Bi3+ and Fe3+ to a wide variety of ligands which include transferrin. Therefore we conclude that the strength of metal ion binding to transferrin is determined more by the ligand donor set than by the size of the ion.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin interacts directly with heterotrimeric G-proteins and can functionally regulate their activity. Recently, a second caveolin gene has been identified and termed caveolin-2. Here, we report the molecular cloning and expression of a third member of the caveolin gene gamily, caveolin-3. Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are approximately 65% identical and approximately 85% similar. A single stretch of eight amino acids (FED-VIAEP) is identical in caveolin-1, -2, and -3. This conserved region may represent a "caveolin signature sequence" that is characteristic of members of the caveolin gene family. Caveolin-3 mRNA is expressed predominantly in muscle tissue-types (skeletal muscle, diaphragm, and heart) and is selectively induced during the differentiation of skeletal C2C12 myoblasts in culture. In many respects, caveolin-3 is similar to caveolin-1: (i) caveolin-3 migrates in velocity gradients as a high molecular mass complex; (ii) caveolin-3 colocalizes with caveolin-1 by immunofluorescence microscopy and cell fractionation studies; and (iii) a caveolin-3-derived polypeptide functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins. Identification of a muscle-specific member of the caveolin gene family may have implications for understanding the role of caveolin in different muscle cell types (smooth, cardiac, and skeletal) as previous morphological studies have demonstrated that caveolae are abundant in these cells. Our results also suggest that other as yet unknown caveolin family members are likely to exist and may be expressed in a regulated or tissue-specific fashion.

A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization ofthese purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as fTishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.Caveolae are plasma membrane specializations (1). Caveolin, a 21-to 24-kDa integral membrane protein, has been identified as a principal component of caveolae membranes in vivo (2, 3). Purification of caveolin-rich membrane domains reveals several distinct classes of signaling molecules (3-6). These include heterotrimeric guanine nucleotide binding proteins (G proteins; a and 13'y subunits), Src-like kinases, protein kinase Ca, and Rap GTPases. Based on these observations, we have proposed (1) that caveolin may function as a scaffolding protein to organize and concentrate inactive signaling molecules within caveolae membranes-for activation by appropriate receptors. This caveolae signaling hypothesis states that "compartmentalization of ceftain cytoplasmic signaling molecules within caveolae could allow efficient and rapid coupling of activated receptors to more than one effector system" (1). In support of this view, inactive G a subunits interact directly with caveolin in a 1:1 stoichiometry-holding them in an inactive conformation (7). Thus, knowledge of the subunit structure of caveolin is important for understanding how caveolin might function to organize or concentrate G a subunits and other signaling molecules within caveolae membranes.In this report, we show that caveolin interacts with itself to form a discrete high molecular mass oligomer. As caveolin also interacts with G a subunits, self oligomerization of caveolin could provide a means for concentrating trimeric G proteins and other caveolin-interacting molecules within caveolae. The existence of caveolin homooligomers complexed with inactive G proteins could explain the observations of Rodbell and colleagues (8), who observed that inactive G proteins exist as high molecular mass oligomeric complexes and that activated G proteins dissociate to monomers. Similarly, activated G proteins fail to interact with recombinant caveolin (7). MATERIALS & METHODSMaterials. Antibodies to carbonic anhydrase IV and glutathione S-transferase (GST) were gifts of W. S. Sly (St. Louis University) and R. Young (Whitehead I...

Caveolae are plasma membrane-attached vesicular organelles. Caveolin-1, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Both caveolae and caveolin are most abundantly expressed in terminally differentiated cells: adipocytes, endothelial cells, and muscle cells. Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-abl and H-ras (G12V); caveolae are absent from these cell lines. However, its remains unknown whether down-regulation of caveolin-1 protein and caveolae organelles contributes to their transformed phenotype.Here, we have expressed caveolin-1 in oncogenically transformed cells under the control of an inducible-expression system. Regulated induction of caveolin-1 expression was monitored by Western blot analysis and immunofluorescence microscopy. Our results indicate that caveolin-1 protein is expressed well using this system and correctly localizes to the plasma membrane. Induction of caveolin-1 expression in v-Abl-transformed and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae as seen by transmission electron microscopy. Consistent with its antagonism of Ras-mediated cell transformation, caveolin-1 expression dramatically inhibited both Ras/MAPK-mediated and basal transcriptional activation of a mitogen-sensitive promoter. Using an established system to detect apoptotic cell death, it appears that the effects of caveolin-1 may, in part, be attributed to its ability to initiate apoptosis in rapidly dividing cells. In addition, we find that caveolin-1 expression levels are reversibly down-regulated by two distinct oncogenic stimuli. Taken together, our results indicate that down-regulation of caveolin-1 expression and caveolae organelles may be critical to maintaining the transformed phenotype in certain cell populations.

Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 skeletal myoblasts in culture. Using differentiated C2C12 skeletal myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, alpha-sarcoglycan, and beta-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.

BackgroundThe complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer.Methods and FindingsPlasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer.ConclusionsOur findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.

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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 A␤ 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 ␣-secretase, an as yet unidentified enzyme that cleaves APP within the A␤ 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, ␣-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 ␣-secretase-mediated proteolysis of APP in vivo.Senile plaques and paired helical filaments are the hallmarks of the brain pathology of Alzheimer's disease (1). The principal component of the senile plaque is the A␤ amyloid peptide, which is composed of 39 -43 amino acid residues. The A␤ amyloid peptide is derived from a full-length precursor protein, termed APP 1 (amyloid precursor protein) (2). Alternate splicing of the APP gene generates at least 10 distinct isoforms; APP 695 is the brain-specific isoform. The A␤ amyloid peptide is generated by the processing of APP with ␤-and ␥-secretases (3). Alternatively, APP is processed by ␣-secretase, which cleaves APP within the A␤ sequence, thereby precluding the formation of A␤ (4). The identities of these secretases remain unknown.In some inherited forms of Alzheimer's disease, point mutations have been identified within the coding sequence of the APP gene (5). These mutations co-segregate with the disease phenotype and cause Alzheimer's disease. Understanding the molecular function and processing of APP is therefore critical to unraveling the molecular basis of Alzheimer's disease.One approach to elucidate the function of APP is to identify APP-interacting proteins. At least five distinct classes of molecules have been identified as APP binding partners as follows: G o (6), Fe 65 (7), X11 protein (8), Fe 65 -like protein (9), and APP-BP1 (10). The APP domain that interacts with G o has been localized to residues His 657 -Lys 676 within the cytoplasmic domain of APP 695 . G o is a brain-specific member of heterotrimeric GTP-binding protein (G-protein) family. The in vivo interaction between APP 695 and G o results in apoptotic cell death (11) and inhibition of cAMP response element trans-activation (12). In contrast, functional consequences of interactions between APP and other binding partners have not yet been described. However, it is likely th...