DIMERIZATION OF TRANSCOBALAMIN-II RECEPTOR - REQUIREMENT OF A STRUCTURALLY ORDERED LIPID BILAYER

Transcobalamin II receptor (TC II-R) exists as a monomer and a dimer o f molecular masses of 62 and 124 kDa in the microsomal and plasma memb ranes, respectively, and in vitro, pure TC II-R monomer dimerizes upon insertion into egg PC/cholesterol (molar ratio, 4:1) liposomes (Bose, S., Seetharam, S., and Seetharam, B. (1995) J. Biol. Chem. 270, 8152- 8157 and Bose, S., Seetharam, S., Hammond, T., and Seetharam, B. (1995 ) Biochem. J. 310, 923-929). The current studies were carried out to d efine the mechanism of TC II-R dimerization. Both the mature TC II-R ( 62 kDa) and the enzymatically deglycosylated TC II-R (45-47 kDa) demon strated optimal association and formed dimers of molecular masses of 9 5 and 124 kDa, respectively, at 22 degrees C when bound to egg PC vesi cles containing at least 10 mol % of cholesterol, Mature TC II-R dimer ized upon insertion into synthetic phosphatidylcholine vesicles of dif ferent fatty acyl chain length (dimyristoyl, dipalmitoyl, and disteroy l phosphatidylcholine) in the absence or the presence of cholesterol a t temperatures below or above their transition temperatures, respectiv ely, Dimerization of TC II-R also occurred with vesicles prepared usin g lipid extract from the plasma but not microsomal membranes, Choleste rol depletion of native intestinal plasma membranes or its enrichment in the microsomal membranes resulted in the in situ conversion of the 124-kDa dimer to the 62-kDa monomer or of the monomer into the dimer f orm, respectively, Treatment of plasma membranes with phospholipase A( 2) resulted in the conversion of the dimer form of the receptor to the monomer form and spin label studies using 1-palmitoyl, 12 doxylsteroy l phosphatidylcholine revealed that interactions of TC II-R with PC ve sicles increased order around the probe, Based on these results we sug gest that dimerization of TC II-R is mediated by its interactions with a rigid more ordered lipid bilayer membrane, is regulated in plasma m embranes by cholesterol levels, and is independent of glycosylation-me diated folding.