High lateral mobility of endogenous and transfected alkaline phosphatase: a phosphatidylinositol-anchored membrane protein.
The lateral mobility of alkaline phosphatase (AP) in the plasma membrane of osteoblastic and nonosteoblastic cells was estimated by fluorescence redistribution after photobleaching in embryonic and in tumor cells, in cells that express AP naturally, and in cells transfected with an expression vector containing AP cDNA. The diffusion coefficient (D) and the mobile fraction, estimated from the percent recovery (%R), were found to be cell-type dependent ranging from (0.58 +/- 0.16) X 10(-9) cm2s-1 and 73.3 +/- 10.5 in rat osteosarcoma cells ROS 17/2.8 to (1.77 +/- 0.51) X 10(-9) cm2s-1 and 82.8 +/- 2.5 in rat osteosarcoma cells UMR106. Similar values of D greater than or equal to 10(-9) cm2s-1 with approximately 80% recovery were also found in fetal rat calvaria cells, transfected skin fibroblasts, and transfected AP-negative osteosarcoma cells ROS 25/1. These values of D are many times greater than "typical" values for membrane proteins, coming close to those of membrane lipid in fetal rat calvaria and ROS 17/2.8 cells (D = [4(-5)] X 10(-9) cm2s-1 with 75-80% recovery), estimated with the hexadecanoyl aminofluorescein probe. In all cell types, phosphatidylinositol (PI)-specific phospholipase C released 60-90% of native and transfection-expressed AP, demonstrating that, as in other tissue types, AP in these cells is anchored in the membrane via a linkage to PI. These results indicate that the transfected cells used in this study possess the machinery for AP insertion into the membrane and its binding to PI. The fast AP mobility appears to be an intrinsic property of the way the protein is anchored in the membrane, a conclusion with general implications for the understanding of the slow diffusion of other membrane proteins.
D000469:Alkaline Phosphatase / Q000502:physiology*; D000818:Animals; D000954:Antigens, Surface / Q000502:physiology; D002117:Calcitriol / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology*; D002478:Cells, Cultured; D003260:Contact Inhibition; D003907:Dexamethasone / Q000494:pharmacology; D008560:Membrane Fluidity / Q000187:drug effects; D008565:Membrane Proteins / Q000502:physiology*; D010006:Osteoblasts / Q000502:physiology; D010716:Phosphatidylinositols / Q000502:physiology*; D051381:Rats; D018800:Thy-1 Antigens; D014162:Transfection; D010738:Type C Phospholipases
D000954:Antigens, Surface; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D018800:Thy-1 Antigens; D003907:Dexamethasone; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D002117:Calcitriol