Alternative promoter usage of the membrane glycoprotein CD36

Background  

CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation.     

Alternative proteolytic processing of platelet membrane glycoprotein IIb

Platelet membrane glycoprotein (GP) IIb-IIIa is a component of a receptor for the adhesive proteins fibrinogen, fibronectin, and von Willebrand factor. GPIIb is initially synthesized as a single-chain polypeptide that is proteolytically processed to yield the two chains of mature GPIIb present on the cell surface. Analysis of the amino acid sequence surrounding the proposed light-heavy chain junction of GPIIb suggests a second potential site following a pair of basic residues 12-15 residues upstream from the reported amino terminus of the light chain. We have utilized anti-peptide antibodies to examine the possibility of alternative cleavage at these two potential sites. Peptide V43 precedes the dibasic sequence and is known to reside in the heavy chain. Peptide V41 contains the sequence between the two potential sites. In immunoblots, anti-V43 reacted only with the heavy chain while anti-V41 reacted only with the light chain. Immunoprecipitation of surface-labeled platelets indicated 97% of the GPIIb light chain contains the V41 sequence while approximately 3% of GPIIb molecules lack the V41 sequence on both the light and heavy chains. These data indicate that GPIIb is primarily cleaved 12-15 amino acids upstream from the reported amino terminus of the light chain while in a minor proportion of GPIIb molecules cleavage occurs at both sites.     

Alternative promoter usage by aldolase A during in vitro myogenesis

Aldolase A in the mouse, as in human and rat, shows tissue-specific variability of message size. In addition, in muscle tissue the mRNA size is also developmentally regulated. In order to determine whether this muscle-specific regulatory mechanism can be reproduced in vitro, we have examined the mRNA species of aldolase A isolated from mouse C2C12 myoblasts and myotubes on Northern blots and by primer extension. We show that aldolase A mRNA increases during in vitro myogenesis; that this induction is accompanied by a change in the message population; and that this change is due to activation of a muscle-specific alternative promoter.     

Isolation and characterization of platelet glycoprotein IV (CD36)

Platelet glycoprotein IV (GPIV, Mr 88,000), which is immunologically related to the leukocyte differentiation antigen CD36, has been isolated from both intact and trypsinized platelet membranes by a series of steps involving (i) phase partitioning in Triton X-114, (ii) ion exchange chromatography on DEAE-cellulose, (iii) lectin affinity chromatography on wheat germ agglutinin-Sepharose, and (iv) size exclusion chromatography on Ultrogel AcA-44. The homogenous product contained 26% carbohydrate (sialic acid, Gal, Man, GalNAc, GlcNAc), of which approximately two-thirds were in alkali-labile O-glycosidic linkages. A rabbit polyclonal antibody raised against purified GPIV gave a single band on immunoblot and on immunoprecipitation from solubilized, 3H-labeled platelet membranes indicating its monospecificity. The antibody gave a strongly positive reaction with platelets on flow cytofluorimetry further confirming the surface location of GPIV. Immunoblotting and flow cytometry also identified GPIV-like molecules on the surface of U937, HEL, and C32 cells but not on HT1080 fibroblasts. Amino acid analysis showed values comparable with those deduced from the cloning data for GPIb, GPIIb, and GPIIIa. Automated Edman degradation allowed the identification of the sequence of the first 36 residues of the NH2-terminal domain. G-X-D-R-N-X-G-L-I-A-G-A-V-I-G-A-V- L-A-V-F-G-G-I-L-M-P-V-G-D-L-P-X-Q-K-F. There are no identifiable homologies between the NH2-terminal domain and other known protein sequences. Following a hydrophilic hexapeptide, there is a hydrophobic sequence of 23 amino acids (underlined) that is of the size and composition expected for a transmembrane domain. Since the NH2-terminal domain lacks tyrosine, but GPIV may be readily iodinated in intact platelets, this suggests that GPIV may have a configuration expressing other extramembranous domains.     

CD36 is a ditopic glycoprotein with the N-terminal domain implicated in intracellular transport

The CD36 receptor sequence predicts two hydrophobic domains located at the N- and C-termini of the protein, but there are conflicting reports as to whether the N-terminal uncleaved leader sequence functions as a transmembrane domain. To investigate the topology of CD36, we generated a panel of mutants lacking either one or both hydrophobic regions and analyzed their folding and transport in COS-7 cells. The N- and the C-terminal hydrophobic regions were both sufficient to anchor CD36 in the membrane, and a FLAG epitope inserted at the N-terminus was located intracellularly. These results indicate that CD36 adopts a ditopic configuration. Accordingly, neither N- nor C-terminal truncation mutants were secreted. Analysis with conformation-specific monoclonal antibodies showed that the N-terminal transmembrane domain truncated molecule was slowly transported through the exocytic pathway and largely accumulated intracellularly. Thus, dual membrane insertion dictates the correct topogenesis and seems to be necessary for efficient folding and intracellular transport.     

Context dependent role of the CD36--thrombospondin--histidine-rich glycoprotein axis in tumor angiogenesis and growth

The angiogenic switch is a promising therapeutic target in cancer. Work by our laboratory and others has described an important endogenous anti-angiogenic pathway mediated by interactions of CD36, a receptor on microvascular endothelial cells, with proteins containing thrombospondin (TSP) type I repeat domains (TSR). Recent studies revealed that circulating Histidine Rich Glycoprotein (HRG) inhibits the anti-angiogenic potential of the CD36-TSR pathway by functioning as a decoy receptor that binds and sequesters TSR proteins. As tumors of different origin display variable expression profiles of numerous targets, we hypothesized that the TSP-CD36-HRG axis regulates vascularization and growth in the tumor microenvironment in a context, or tumor type, dependent manner. Growth of Lewis Lung Carcinoma (LL2) and B16F1 Melanoma tumor cell implants in syngeneic wild type (WT), hrg, or cd36 null mice were used as a model to interrogate this signaling axis. LL2 tumor volumes were greater in cd36 null mice and smaller in hrg null mice compared to WT. Immunofluorescent staining showed increased vascularity in cd36 null vs. WT and WT vs. hrg null mice. No differences in tumor growth or vascularity were observed with B16F1 implants, consistent with lack of expression of TSP-1 in B16F1 cells. When TSR expression was induced in B16F1 cells by cDNA transfection, tumor growth and vascularity were similar to that seen with LL2 cells. These data show a role for CD36-mediated anti-angiogenic activity in the tumor microenvironment when TSR proteins are available and demonstrate that HRG modulates this activity. Further, they suggest a mechanism by which tumor microenvironments may regulate sensitivity to TSR containing proteins.     

Sense and antisense cDNA transfection of CD36 (glycoprotein IV) in melanoma cells. Role of CD36 as a thrombospondin receptor.

Thrombospondin (TSP) is a multifunctional matrix and platelet glycoprotein that interacts with cell surfaces and may play a role in mediating cell adhesion, platelet aggregation, platelet-monocyte interactions, cell proliferation, angiogenesis, tumor metastasis, and protease generation. To clarify and confirm the function of CD36 (glycoprotein IV) as a TSP receptor, we now describe a transfected cell model using human melanoma cells genetically manipulated by sense or antisense cDNA transfection to express either high or near zero levels of CD36. Surface expression was confirmed by flow cytometry with monoclonal anti-CD36 IgG and quantified by measuring radiolabeled antibody binding. Bowes melanoma cells, which in their wild type did not express CD36 and did not bind radiolabeled TSP, when transfected with the sense construct bound TSP in a 1:1 stoichiometric ratio with CD36 expression. Conversely, C32 melanoma cells, which in their wild type expressed high levels of CD36 and bound radiolabeled TSP at a 1:1 stoichiometric ratio, did not express CD36 and did not bind TSP when transfected with an antisense construct. In addition, transfected Bowes cells and wild type C32 cells, unlike wild type Bowes cells, adhered to activated platelets in a TSP-dependent manner. These data, i.e. the gain of function with sense cDNA transfection and loss of function with antisense transfection, strongly support the TSP receptor function of CD36. The distribution of this protein in vascular cells and tissues and observations that it may participate in signal transduction events suggest that TSP-CD36 interactions may play a role in mediating some of the pathophysiological processes associated with TSP.     

Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion

The role of glycoprotein IV (GPIV) in platelet activation processes has been examined by several different approaches: (i) Fab fragments of a monospecific polyclonal antibody to purified platelet GPIV (approximately 20 micrograms/ml) completely inhibited platelet shape change, aggregation, and secretion induced by collagen. Aggregation and secretion by ADP (but not shape change) and by epinephrine were also inhibited, but there was no effect on platelet activation induced by thrombin, arachidonate, or ionophore A23187. (ii) Purified GPIV was able to compete completely with membrane-bound GPIV to inhibit platelet activation induced by collagen, including shape change, but not in activation induced by any of the other platelet agonists. 50% inhibition of collagen-induced activation and secretion were obtained at GPIV concentrations of approximately 10 nM (1 micrograms/ml). (iii) Purified GPIV bound rapidly and reversibly to collagen Type I fibrils, and binding was not inhibited by adhesive proteins such as denatured collagen, fibronectin, fibrinogen, or von Willebrand factor. The direct binding of purified GPIV to collagen Type I fibrils fit best to a single site model with Kd 0.34 +/- 0.10 nM. (iv) Using a microtiter assay, platelet adhesion to collagen was shown to be inhibited by Fab fragments of monospecific polyclonal anti-GPIV antibodies, but adhesion to other adhesive proteins was unaffected. (v) When anti-GPIV was added at various times during adhesion the time dependence of inhibition was seen to be biphasic. Anti-GP antibody was able to reverse adhesion that occurred within the first 5-8 min and to inhibit adhesion occurring thereafter. These results demonstrate that GPIV mediates the early stages of platelet recognition by and attachment to collagen but that there may be a second GPIV-independent mechanism that mediates the subsequent anchorage of these adherent platelets.     

CD44v10: an antimetastatic membrane glycoprotein for pancreatic cancer

AIMS: The aims of this study were 1) to investigate the mRNA pattern of CD44 variants in three primary (MIA PaCa 2, PANC-1, PSN-1) and two metastatic (CAPAN-1, SUIT-2) pancreatic cancer (PC) cell lines; 2) to ascertain whether the genetic transfer of CD44s and CD44v10 modifies the adhesion of PC cells to the extracellular matrix (ECM) in vitro and their metastatic behavior in vivo. METHODS: CD44 mRNA analysis was done by means of RT-PCR. Adhesion to ECM the was assessed using coated microtiter plates. For the study of CD44v10 insertion in the CAPAN-1 line, liposome-mediated DNA transfer was used. SCID mice were employed for in vivo experiments. RESULTS: CD44v10 mRNA was not expressed by the CAPAN-1 nor by four of the six SUIT-2-derived clones. The stable expression of CD44v10 by modified CAPAN-1 significantly enhanced fibronectin adhesion. Mice without either liver or pancreatic metastases were more frequently found among the animals injected with modified (CD44v10 expressing) than with non-modified CAPAN-1. CONCLUSIONS: 1) It is possible to differentiate between metastatic and non-metastatic PC cells on the basis of CD44v10 expression; 2) CD44v10 seems to be involved in mediating fibronectin adhesion in vitro and in counteracting metastases in vivo.     

Polysialic acid in human milk. CD36 is a new member of mammalian polysialic acid-containing glycoprotein

The neural cell adhesion molecule and the voltage-sensitive sodium channel alpha-subunit are the only two molecules in mammals known to be modified by alpha-2,8-linked polysialic acid (polySia). We found a new polySia-containing glycoprotein in human milk and identified it as CD36, a member of the B class of the scavenger receptor superfamily. The polySia-containing glycan chain(s) were removed by alkaline treatment but not by peptide:N-glycanase F digestion, indicating that milk CD36 contained polySia on O-linked glycan chain(s). Polysialylation of CD36 occurs not only in human milk but also in mouse milk. However, CD36 in human platelets is not polysialylated. PolySia CD36 is secreted in milk at any lactation stage and reaches peak level at 1 month after parturition. Thus, it is suggested that polySia of milk CD36 is significant for neonatal development in terms of protection and nutrition.     

CD36 and atherosclerosis

CD36 has been associated with diverse normal and pathologic processes. These include scavenger receptor functions (uptake of apoptotic cells and modified lipid), lipid metabolism and fatty acid transport, adhesion, angiogenesis, modulation of inflammation, transforming growth factor-beta activation, atherosclerosis, diabetes and cardiomyopathy. Although CD36 was identified more than 25 years ago, it is only with the advent of recent genetic technology that in-vivo evidence has emerged for its physiologic and pathologic relevance. As these in-vivo studies are expanded, we will gain further insight into the mechanism(s) by which CD36 transmits a cellular signal, and this will allow the design of specific therapeutics that impact on a particular function of CD36.     

Conformation of an endogenous ligand in a membrane bilayer for the macrophage scavenger receptor CD36

Phagocytic removal of aged or oxidatively damaged cells and macromolecules is an indispensable homeostatic function of the innate immune system. A structurally conserved family of oxidized phospholipids that serve as endogenous high-affinity ligands for the macrophage scavenger receptor CD36 (oxPC(CD36)) was recently identified. Enriched within atherosclerotic plaque and senescent cell membranes, oxPC(CD36) promote the uptake of oxidized lipoproteins and cell membranes by macrophages when present at only a few molecules per particle. How macrophages recognize oxPC(CD36) within cellular membranes and lipoprotein surfaces remains unknown. Herein, we deduce the conformation of oxPC(CD36) near the hydrophobic-hydrophilic interface within membrane bilayers by determining multiple critical internuclear distances using nuclear Overhauser enhancement spectroscopy. The molecular model reveals a unique conformation for oxPC(CD36) within bilayers whereby the distal end of the sn-2 acyl chain harboring the structurally conserved CD36 recognition motif protrudes into the aqueous phase. The remarkable conformation elucidated for oxPC(CD36) produces a surface accessible phagocytic "eat me signal" to facilitate senescent cell and oxidized lipoprotein recognition by the scavenger receptor CD36 as part of its immune surveillance function.     

The antiangiogenic effect of thrombospondin-2 is mediated by CD36 and modulated by histidine-rich glycoprotein.

Thrombospondins-1 and -2 (TSP-1, TSP-2) are matricellular glycoproteins with potent antiangiogenic activity. We have previously shown that the antiangiogenic activity of TSP-1 is mediated by the interaction of the type I repeats (TSR) with the receptor CD36, although other domains of TSP-1 have also been implicated. We now show that the antiangiogenic activity of TSP-2, which contains three TSRs but, unlike TSP-1, lacks the capacity to activate TGF-beta, is similarly dependent on CD36. Using the corneal pocket assay we found that TSP-2 did not inhibit bFGF-induced angiogenesis in CD36 null mice. We then demonstrated that (125)[I]-TSP-2 bound to murine macrophages and that binding was diminished by 70% by anti-CD36 antibody or by using cells from CD36 null animals. Solid-phase binding studies revealed that (125)[I]-TSP-2 bound to CD36/glutathione-S-transferase (GST) fusion proteins encoding the region spanning amino acids 93-120, but not amino acids 298-439. This 93-120 amino acid region, previously identified as the TSP-1 binding site, is homologous to domains on other TSP binding proteins, such as LIMP-2 and histidine-rich glycoprotein (HRGP). Finally, we showed with an immunoabsorbent binding assay that TSP-2 bound HRGP with high affinity and that HRGP blocked the antiangiogenic activity of TSP-2, acting like a "decoy" receptor. These data suggest that modulation of the TSR/CD36 system may play an important role in the regulation of the angiogenic "switch," and may provide a target for therapeutic interventions.