Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure.

Caveolae are indentations of the plasma membrane of mammalian cells that are approximately 50–100 nm in diameter (1). These microdomains are intimately involved in numerous cellular functions ranging from signal transduction to lipid recycling (2). Caveolae are ubiquitous, and can be found in most terminally differentiated cell types, notably smooth muscle cells and adipocytes (1). Caveolae are primarily comprised of a protein called caveolin, which is critical for the formation of caveolae. It is estimated that approximately 100–200 caveolin proteins are present in a single caveola, and studies have shown that when the caveolin gene is silenced, caveolae disappear from the surface of cells (3, 4). Caveolin is an integral membrane protein, and approximately 50% of the polypeptide chain is predicted to interact with the plasma membrane. Glycosylation mapping, limited proteolysis, and cell surface biotinylation studies have demonstrated that the N- and C-termini of caveolin reside on the cytosolic side of the membrane (5, 6, 7). The transmembrane region of caveolin is approximately 33 amino acids in length, which is significantly longer than the 20–25 amino acids required to span the plasma membrane. In addition, there are no soluble amino acids present in the transmembrane region of caveolin that would facilitate a polytopic orientation. Taken together, these findings have led to the postulation that caveolin forms a horseshoe-like loop in the membrane (Figure 1). The horseshoe-like loop conformation is believed to facilitate the high degree of membrane curvature observed in caveolae (4). Furthermore, the membrane curvature is believed to be stabilized by the formation of high molecular weight caveolin oligomers. Figure 1 A) Cartoon of caveolin-1 topology. The transmembrane domain is highlighted in red. The zigzag lines represent sites of cysteine palmitoylation. Caveolin has been closely linked to a number of disease states including heart disease, Alzheimer’s, and cancer. Specifically, a naturally occurring point mutation (P132L) in the caveolin-1 gene is present in approximately 16% of breast cancers, and has been closely linked to increased recurrence and metastasis (8, 9). In addition, this mutant appears to be functionally different from the wild-type as it is retained in the Golgi apparatus of cells and does not get transported to its final destination in the plasma membrane. The expression of the P132L mutant results in the elimination of caveolae from the surface of cells (10). Interestingly, the P132L mutant appears to behave in a dominant negative manner: When both the P132L mutant and wild-type caveolin are co-expressed, both become localized to the Golgi, which is a fate common to misfolded proteins (10). Furthermore, a study by Bonuccelli et al demonstrates that when caveolin-1 P132L is expressed in Met-1 cells, it acts as a loss-of-function mutation and promotes recurrence of breast cancer (8). In this report, we examine both the oligomeric and secondary structural changes that occur in the caveolin-1 P132L mutant versus wild-type caveolin-1. Using gel filtration chromatography and sedimentation equilibrium analytical ultracentrifugation we show that the P132L mutant is dimeric compared to the wild-type which is monomeric. Using various truncated constructs, we have isolated the dimerization region to the transmembrane domain which is where the P132L mutation resides. In addition, we show that substitution of proline 132 with 6 other nonpolar amino acids does not disrupt dimerization, which reveals the importance of proline at position 132. Using chemical shift indexing NMR analysis we found that the putative re-entrant helix of the P132L mutant is extended by 4 additional amino acids which results in a longer α-helix when compared to the wild-type. Furthermore, the NMR studies reveal that the P132L point mutation results in only a localized change to the transmembrane region of caveolin rather than a global conformation change.