Characterization of the molecular architecture of human caveolin-3 and interaction with the skeletal muscle ryanodine receptor.
BACKGROUND
Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca(2+) homeostasis.


RESULTS
Caveolin-3 forms a disc-shaped nonamer that binds the Ca(2+)-release channel, RyR1.


CONCLUSIONS
Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer.


CONCLUSIONS
First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane. Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ~16.5 nm in diameter and ~ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.
School of Biomedicine, Cardiovascular Group, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9NT, UK.
Gareth
G
Whiteley
Richard F
RF
Collins
Ashraf
A
Kitmitto
D002118:Calcium / Q000378:metabolism; D021941:Caveolae / Q000737:chemistry / Q000378:metabolism; D051244:Caveolin 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D051244:Caveolin 3; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium