PTH-induced internalization of a type IIa Na/P-i cotransporter in OK-cells

Regulatory phenomena in brush border membrane sodium/phosphate (Na/P-i) cot ransport are directly related to the type IIa Na/P-i-cotransporter and can be analyzed in opossum kidney cells (OK-cells). Parathyroid hormone (PTH) l eads to a decreased expression of the type IIa Na/P-i-cotransporter protein at the apical cell surface. To provide evidence for PTH-induced membrane r etrieval of the cotransporter protein we labeled OK-cell surface membrane p rotein NH2-groups with N-hydroxysuccinimide bound via a disulfide bond to b iotin (NHS-SS-biotin) prior to or after treatment with PTH. Biotinylated tr ansporters can be detected by streptavidin precipitation and Western blotti ng using type IIa Na/P-i-cotransporter specific antibodies. To detect only internalized biotinylated transporters biotin located at the cell surface w as removed ("stripped") by disulfide bond splitting reagents under reducing conditions. Neither biotinylation per se, nor "stripping" interfered with PTH-induced inhibition of Na/P-i-cotransport activity. The internalization of the transporter was highly increased in response to PTH treatment. The d ata document that the first step in PTH regulation is internalization of th e type IIa Na/P-i-cotransporter protein from the apical membrane.