Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae.Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein Thyl, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.Caveolae are specialized domains of the plasma membrane present in most mammalian cell types. While the function of these structures remains controversial, their morphology has been studied in great detail (1). Caveolae generally appear as 50-to 90-nm vesicular invaginations of the plasma membrane with a characteristic bulb shape. Clusters of connected caveolae resembling bunches of grapes are often seen in adipocytes, endothelial cells, and fibroblasts. In contrast to clathrin-coated pits, caveolae do not show a well-defined coat by conventional transmission electron microscopy. However, filamentous material arranged in spiral or striated arrays on the cytoplasmic side of caveolae has been revealed by other electron microscopy techniques (2, 3) and is generally referred to as the caveolar coat. VIP21-caveolin, an integral membrane protein originally identified as a substrate for v-src (4) and a component of Golgi-derived transport vesicles (5), was localized in caveolae by immunogold staining and proposed to play a structural role (6, 7). We have previously reported that the absence of detectable VIP21-caveolin correlates with the lack of caveolae in a lymphocyte cell line (8). Similar results were obtained in N2a neuroblastoma cells (9). Here we express VIP21-caveolin in our model lymphocyte cell line and analyze the plasma membrane morphology by electron microscopy. Consistent with its proposed structural role, expression of VIP21-caveolin induces formation of caveolae.

Abstract.We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.

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In an effort to define the actual function of the promiscuous putatively silent chemokine receptor D6, transfectants were generated in different cell types. Engagement of D6 by inflammatory CC chemokines elicited no calcium response nor chemotaxis, but resulted in efficient agonist internalization and degradation. Also in lymphatic endothelium, where this receptor is expressed in vivo, D6 did not elicit cellular responses other than ligand internalization and degradation. In particular, no evidence was obtained for D6-mediated transcytosis of chemokines in the apical-to-basal or basal-to-apical directions. These results indicate that D6 acts as an inflammatory chemokine scavenging nonactivatory decoy receptors and suggest that in lymphatic vessels D6 may function as a gatekeeper for inflammatory CC chemokines, by clearing them and preventing excessive diffusion via afferent lymphatics to lymph nodes.