SUMMARYIt has recently been shown that several members of the tetraspan superfamily, including CD9 and CD63, associate with each other and with ␤ 1 integrins. In this study, we examined the distribution of a recently identified tetraspan, PETA-3 (CD151), and of CD9, CD63, ␣ 5 ␤ 1, and the integrin ␤ 1 chain in normal human tissues by the indirect immunoperoxidase and alkaline phosphatase-anti-alkaline phosphatase techniques. PETA-3 showed a broad distribution and was expressed by endothelium, epithelium, Schwann cells, and dendritic cells and by skeletal, smooth, and cardiac muscle. Expression in skin was mostly restricted to the basal cells of the epidermis and was downregulated on differentiation. In the small intestine, PETA-3 was expressed by crypt and villous enterocytes with a mostly basolateral distribution, but was not detectable on the brush border. CD9 was expressed on the plasma membrane of enterocytes in crypts and at the bases of the villi whereas CD63 demonstrated a unique granular appearance concentrated in the apical cytoplasm below the brush border. The findings of this study show co-localization of PETA-3 with CD9, CD63, ␣ 5 ␤ 1, and ␤ 1 in particular tissues, demonstrating that tetraspan/integrin complexes may occur. However, the lack of co-localization of these antigens in other tissues also implies distinct roles for these molecules. ( J Histochem Cytochem 45:515-525, 1997 )

CD151 (PETA-3/SFA-1) is a member of the transmembrane 4 superfamily (TM4SF) of cell-surface proteins and is expressed abundantly both on the cell surface and in intracellular membranes by the haemopoietic cell lines M07e, HEL and K562. In the presence of mild detergent (CHAPS), CD151 co-immunoprecipitated with integrin alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha IIb beta 3. The association of CD151 with alpha 4 beta 1 and alpha 5 beta 1 seemed to be constitutive, as it was not modified by treatment of M07e cells with cytokines that regulate integrin function by 'inside-out' signalling. CD151 also associated with other tetraspans in an apparently cell-type-specific fashion, as defined by its co-precipitation with CD9, CD63 and CD81 from M07e cells, but not from K562 cells, which express similar levels of these proteins. F(ab')2 fragments of monoclonal antibodies (mAbs) against CD151 caused homotypic adhesion of HEL and K562 cells that was dependent on energy and cytoskeletal integrity and was augmented in the presence of RGDS peptides. The adhesion was not blocked by function-inhibiting mAbs against beta 1 or beta 3 integrins, suggesting that cell-cell adhesion was not mediated by the binding of integrin to a cell-associated ligand. Furthermore, mAb CD151 did not affect adhesion of the cells to fibronectin, laminin, collagen or fibrinogen, which are ligands for alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha IIb beta 3 integrins. Taken together, these results indicate that the ligation of CD151 does not induce the up-regulation of integrin avidity, but might act as a component of integrin signalling complexes.

Tail-interacting 47-kDa protein (TIP47) binds the cytoplasmic domains of the cation-dependent (CD) and cation-independent (CI) mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the Golgi complex. TIP47 recognizes a phenylalanine-tryptophan signal in the CD-MPR. We show here that TIP47 interaction with the 163-residue CI-MPR cytoplasmic domain is highly conformation dependent and requires CI-MPR residues that are proximal to the membrane. CI-MPR cytoplasmic domain residues 1-47 are dispensable, whereas residues 48 -74 are essential for high-affinity binding. However, residues 48 -74 are not sufficient for high-affinity binding; residues 75-163 alone display weak affinity for TIP47, yet they contribute to the presentation of residues 48 -74 in the intact protein. Independent competition binding experiments confirm that TIP47 interacts with the membrane-proximal portion of the CI-MPR cytoplasmic domain. TIP47 binding is competed by the binding of the AP-2 clathrin adaptor at (and near) residues 24 -29 but not by AP-1 binding at (and near) residues 160 -161. Finally, TIP47 appears to recognize a putative loop generated by the sequence PPAPRPG and other hydrophobic residues in the membrane-proximal domain. Although crystallography will be needed to define the precise interaction interface, these data provide an initial structural basis for TIP47-CI-MPR association.

SUMMARY:The new mAb UA009 recognizes an antigen expressed by microvascular endothelium, by lymphatic endothelium, and by some epithelia in a number of organs, including the small intestine, lactating mammary gland, kidney, lung, sebaceous glands, and circumvallate papillae of the tongue. This antigen is also expressed abundantly in the splenic red pulp and marginal zone and by monocytes, macrophages, and erythrocytes (but not by platelets). Among tissues that store or metabolize fatty acids, the antigen is expressed by adipocytes, cardiomyocytes, and red skeletal muscle. Importantly, it is expressed by steroidogenic cells in the adrenal gland, testis, and ovary, whereas in the liver it is expressed by hepatocytes in a pattern that is dependent on gender and genetic background. mAb UA009 immunoprecipitated a mol wt 85-kDa surface protein from detergent extracts of hepatocytes from Dark Agouti female rats. The N-terminal amino acid sequence of this protein was identical to fatty acid translocase (FAT), the rat cluster of differentiation 36 (CD36) ortholog. The mAb also reacted with COS-7 cells transfected with cDNA encoding FAT. cDNAs encoding a CD36/FAT-like polypeptide were prepared from both liver and heart RNA by RT-PCR. The nucleotide sequences obtained from these cDNAs (Dark Agouti rats) revealed identity and 99% similarity, respectively, with the published sequences of Cd36/Fat in rats of the Wistar and Sprague-Dawley strains. The absence of the UA009 antigen in CD36/FAT-deficient SHR/N rats confirmed the identity of the UA009 antigen and CD36/FAT. We suggest that CD36/FAT might function in the liver as a sex-regulated accessory molecule, either in reverse cholesterol transport and/or in fatty acid uptake. (Lab Invest 2003, 83:317-332).

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