IMMUNODETECTION OF A TYPE-III SODIUM-DEPENDENT PHOSPHATE COTRANSPORTER IN TISSUES AND OK CELLS

Polyclonal antibodies were raised in rabbits against a 14-amino acid p ortion of the gibbon ape leukemia virus human membrane receptor Glvr-1 . This epitope also contained seven amino acids common to the receptor for the amphotropic murine retrovirus Ram-1. Antibody specificity and molecular size of Glvr-1/Ram-1-related proteins were assayed by Weste rn blot. Using a standard Laemmli buffer system, under reducing condit ions, a single band of approximately 85 kDa (designated p85) was immun odetected in membranes prepared from opossum kidney (OK) cells and in brain membranes from rat, rabbit and hamster. In mouse brain, p85 as w ell as a protein of 70-72 kDa were immunodetected. This protein was al so present in several other mouse tissues. Limited proteolysis of p85 and the 70-72kDa-protein from mouse yielded similar peptide fragments, suggesting that both proteins are related. Fragments of the same mole cular masses were also detected in OK cell membranes following proteol ysis, showing that p85 in both models (mouse brain and OK cell) share a similar sequence. p85 is not N-glycosylated since an assay using end oglycosidase F/N-glycosidase F did not alter the electrophoretic mobil ity of p85. We also observed that regulation of phosphate transport by incubating OK cells without any phosphate or by PTH treatment occurs without any changes in the amount of p85, In conclusion, these data de monstrate for the first time a Western blot detection of a type III ph osphate transporter using polyclonal antibodies. They also suggest tha t, conversely to type I and type II phosphate transporters which are l ocalized in the kidney, this third type of transporter is ubiquitous a nd probably absorbs the readily available phosphate from interstitial fluid for normal cellular functions in many species and tissues, servi ng as a housekeeping Na+/P-i cotransport system. This is also the firs t report showing that p85 is not regulated in the same manner as type II phosphate transporters. (C) 1998 Elsevier Science B.V.