CHARACTERIZATION OF CAVEOLIN-RICH MEMBRANE DOMAINS ISOLATED FROM AN ENDOTHELIAL-RICH SOURCE - IMPLICATIONS FOR HUMAN-DISEASE

Caveolae are 50-100-nm membrane microdomains that represent a subcompa rtment of the plasma membrane. Previous morphological studies have imp licated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial. cells, (b) the up take of small molecules via a process termed potocytosis involving GPI -linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartme ntalization of certain signaling molecules, including G-protein couple d receptors. Caveolin, a 22-kD integral membrane protein, is an import ant structural component of caveolae that was first identified as a ma jor v-Src substrate in Rous sarcoma virus transformed cells. This find ing initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured c ells. To facilitate biochemical manipulations, we have applied this pr ocedure to lung tissue- an endothelial and caveolin-rich source-allowi ng large scale preparation of these complexes. These membrane domains retain similar to 85% of caveolin and similar to 55% of a GPI-linked m arker protein, while they exclude greater than or equal to 98% of inte gral plasma membrane protein markers and greater than or equal to 99.6 % of other organelle-specific membrane markers tested. Characterizatio n of these complexes by micro-sequencing and immune-blotting reveals k nown receptors for modified forms of LDL (scavenger receptors: CD 36 a nd RAGE), multiple GPI-linked proteins, an anion transporter (plasma m embrane porin), cytoskeletal elements, and cytoplasmic signaling molec ules-including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1-the Ras tumor suppr essor protein, Rap 2, and TC21). At least a fraction of the actin in t hese complexes appeared monomeric (G-actin), suggesting that these dom ains could represent membrane bound sites for microfilament nucleation /assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis fo r evaluating these interactions in vivo.