Affinity-purification and characterization of caveolins from the brain: differential expression of caveolin-1, -2, and -3 in brain endothelial and astroglial cell types.
Caveolins 1, 2 and 3 are the principal protein components of caveolae organelles. It has been proposed that caveolae play a vital role in a number of essential cellular functions including signal transduction, lipid metabolism, cellular growth control and apoptotic cell death. Thus, a major focus of caveolae-related research has been the identification of novel caveolins, caveolae-associated proteins and caveolin-interacting proteins. However, virtually nothing is known about the expression of caveolins in brain tissue. Here, we report the purification and characterization of caveolins from brain tissue under non-denaturing conditions. As a final step in the purification, we employed immuno-affinity chromatography using rabbit polyclonal anti-caveolin IgG and specific elution at alkaline pH. The final purified brain caveolin fractions contained three bands with molecular masses of 52 kDa, 24 kDa and 22 kDa as visualized by silver staining. Sequencing by ion trap mass spectrometry directly identified the major 24-kDa component of this hetero-oligomeric complex as caveolin 1. Further immunocyto- and histochemical analyses demonstrated that caveolin 1 was primarily expressed in brain endothelial cells. Caveolins 2 and 3 were also detected in purified caveolin fractions and brain cells. The cellular distribution of caveolin 2 was similar to that of caveolin 1. In striking contrast, caveolin 3 was predominantly expressed in brain astroglial cells. This finding was surprising as our previous studies have suggested that the expression of caveolin 3 is confined to striated (cardiac and skeletal) and smooth muscle cells. Electron-microscopic analysis revealed that astrocytes possess numerous caveolar invaginations of the plasma membrane. Our results provide the first biochemical and histochemical evidence that caveolins 1, 2 and 3 are expressed in brain endothelial and astroglial cells.
Ikezu
T
T
Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Ueda
H
H
Trapp
B D
BD
Nishiyama
K
K
Sha
J F
JF
Volonte
D
D
Galbiati
F
F
Byrd
A L
AL
Bassell
G
G
Serizawa
H
H
Lane
W S
WS
Lisanti
M P
MP
Okamoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001921:Brain / Q000166:cytology; D001923:Brain Chemistry / Q000502:physiology*; D002417:Cattle; D051242:Caveolin 1; D051243:Caveolin 2; D051244:Caveolin 3; D022461:Caveolins; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004730:Endothelium, Vascular / Q000378:metabolism*; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D008565:Membrane Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D008566:Membranes / Q000737:chemistry; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley
C495537:Cav1 protein, rat; C495549:Cav3 protein, rat; D051242:Caveolin 1; D051243:Caveolin 2; D051244:Caveolin 3; D022461:Caveolins; D007074:Immunoglobulin G; D008565:Membrane Proteins