MUTATIONAL ANALYSIS OF THE PROPERTIES OF CAVEOLIN-1 - A NOVEL ROLE FOR THE C-TERMINAL DOMAIN IN MEDIATING HOMO-TYPIC CAVEOLIN-CAVEOLIN INTERACTIONS

Caveolin is a principal structural component of caveolae membranes in vivo, Recently, a family of caveolin-related proteins has been identif ied; caveolin has been retermed caveolin-1. Caveolin family members sh are three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes . Here, we have used a deletion mutagenesis approach as a first step t oward understanding which regions of caveolin-1 contribute to its unus ual properties, Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1 Delta C) or the N-terminal domain (Cav-1 Delta N); these mutants were compared with the behavior of ful l-length caveolin-1 (Cav-1FL) expressed in parallel, Our results show that the N-terminal domain and membrane spanning segment are sufficien t to form high molecular mass oligomers of caveolin-1. However, a comp lete caveolin-1 molecule is required for conveying detergent insolubil ity and incorporation into low density Triton-insoluble complexes, The se data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an un known role for the C-terminal domain in this process, To better unders tand the role of the C-terminal domain, this region of caveolin-1 (res idues 135-178) was expressed as a glutathione S-transferase fusion pro tein in Escherichia coli, Purified recombinant glutathione S-transfera se-C-Cav-1 was found to stably interact with full-length caveolin-1 bu t not with the two caveolin-1 deletion mutants, These results suggest that the C-terminal domain interacts with both the N-terminal and C-te rminal domains of an adjacent caveolin-1 homo-oligomer, This appears t o be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3, Homo-typic interaction of the C-terminal domain with a n adjacent homo-oligomer could provide a mechanism for clustering cave olin-1 homooligomers while excluding other caveolin family members. Th is type of lateral segregation event could promote caveolae membrane f ormation and contribute to the detergent insolubility of caveolins-1, -2, and -3.