title: Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure.
abstract: Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation in vivo and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation, we found that a fully functional construct of caveolin-1 (Cav1(62-178)) was a monomer in dodecylphosphocholine micelles. In contrast, the P132L mutant of Cav1(62-178) was dimeric. To explore the dimerization of the P132L mutant further, various truncated constructs (Cav1(82-178), Cav1(96-178), Cav1(62-136), Cav1(82-136), Cav1(96-136)) were prepared which revealed that oligomerization occurs in the transmembrane domain (residues 96-136) of caveolin-1. To characterize the mutant structurally, solution-state NMR experiments in lyso-myristoylphosphatidylglycerol were undertaken of the Cav1(96-136) P132L mutant. Chemical shift analysis revealed that, compared to the wild-type, helix 2 in the transmembrane domain was lengthened by four residues (wild-type, residues 111-129; mutant, residues 111-133), which corresponds to an extra turn in helix 2 of the mutant. Lastly, point mutations at position 132 of Cav1(62-178) (P132A, P132I, P132V, P132G, P132W, P132F) revealed that no other hydrophobic amino acid can preserve the monomeric state of Cav1(62-178), which indicates that proline 132 is critical in supporting proper caveolin-1 behavior.
authors.affiliation: Department of Chemistry, Lehigh University, 6 E. Packer Ave, Bethlehem, Pennsylvania 18015, USA.
authors.forename: Monica D
authors.initials: MD
authors.lastname: Rieth
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Lee
authors.forename: Kerney Jebrell
authors.initials: KJ
authors.lastname: Glover
mesh_terms: D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D014461:Ultracentrifugation
chemical_list: D051242:Caveolin 1; D011392:Proline