TRANSMEMBRANE DOMAIN OF CD44 IS REQUIRED FOR ITS DETERGENT INSOLUBILITY IN FIBROBLASTS

The hyaluronan receptor CD44 is an abundant glycoprotein expressed on a variety of different cell types, In fibroblasts a significant portio n of receptor molecules remain in the detergent-insoluble fraction aft er Triton X-100 extraction. Detergent insolubility of these CD44 molec ules has been interpreted to reflect their association with the cytosk eleton, In this study we examined the structural features of CD44 requ ired for its Triton X-100 insolubility in murine fibroblasts. We expre ssed in L cells the wild-type hematopoietic form of CD44, a mutant CD4 4 lacking the cytoplasmic domain, and two mutant CD44 molecules with s ubstituted transmembrane domains, Immunofluorescence and cell surface iodination were performed and the detergent extraction profile of the transfected CD44 molecules was determined, No difference in detergent solubility was observed between wild-type and tailless mutant-transfec ted molecules, However, both CD44 mutants with a heterologous transmem brane domain, derived from either the CD3 zeta chain or CD45, were com pletely soluble in Triton X-100. These results demonstrate that the tr ansmembrane region but not the cytoplasmic domain of CD44 is required for the detergent-insolubility in these cells, No obvious colocalizati on of CD44 and actin stress fibers was observed before or after treatm ent with cytochalasin D, and no change in the detergent extraction pro file of wild-type and mutant CD44 molecules was effected by cytochalas in D, In equilibrium density sucrose gradients the Triton-insoluble CD 44 component was found in the low density fractions, indicating an ass ociation with Triton X-100-insoluble lipids, Together, these results s uggest that Triton X-100 insolubility of a fraction of the CD44 molecu les in fibroblasts is not the result of interaction with the actin-bas ed cytoskeleton but rather due to an association with Triton-insoluble lipids, which is, in turn, dependent on the transmembrane domain of C D44.