Modulation of Phosphate Uptake and Amphotropic Murine Leukemia Virus Entry by Posttranslational Modifications of PIT-2

Abstract

PIT-2 is a type III sodium phosphate cotransporter and the receptor for amphotropic murine leukemia viruses. We have investigated the expression and the functions of a tagged version of PIT-2 in CHO cells. PIT-2 remained equally abundant at the cell surface within 6 h following variation of the phosphate supply. In contrast, the efficiency of phosphate uptake and retrovirus entry was inversely related to the extracellular phosphate concentration, indicating that PIT-2 activities are modulated by posttranslational modifications of cell surface molecules induced by phosphate. Conformational changes of PIT-2 contribute to both activities, as shown by the inhibitory effect of sulfhydryl reagents known as inhibitors of type II cotransporters. A physical association of PIT-2 with actin was demonstrated. Modifications of the actin network were induced by variations of the concentrations of extracellular phosphate, cytochalasin D, or lysophosphatidic acid. They revealed that the formation of actin stress fibers determines the cell surface distribution of PIT-2, the internalization of the receptor in response to virus binding, and the capacity to process retrovirus entry. Thus, the presence of PIT-2 at the cell surface is not sufficient to ensure phosphate transport and susceptibility to amphotropic retrovirus infection. Further activation of cell surface PIT-2 molecules is required for these functions.

Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

“…The present results showing a caveola-mediated endocytic entry via Pit2 in CHO K1 cells is strengthened by the fact that disturbance of the actin cytoskeleton impaired A-MLV infection of CHO K1 cells (45) as caveolae are known to be anchored to the actin cytoskeleton and disruption of actin assembly blocks caveola-mediated endocytosis (54).…”

Apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors

“…Since Pit2 is a sodium-dependent phosphate transporter, we used inorganic phosphate as a competitive inhibitor of Pit2 binding as described by others. 19 When 40 mM NaH 2 PO 4 was included in the solution with the apically applied MuLV, binding was inhibited (third bar). In contrast, 40 mM NaHCO 4 had no such inhibitory effect on binding (fourth bar).…”

“…Furthermore, these studies indicate that the amphotropic MuLV vector binds to the apical surface of airway epithelia and that this binding is reduced in the presence of NaH 2 PO 4 , suggesting it is mediated by Pit2. 19 Therefore the limitation for apical Gene Therapy gene transfer with amphotropic retrovirus is more than the simple absence of the appropriate cellular receptor. Increasing Pit2 expression in airway epithelial cells with an adenoviral vector did not enhance apical gene transfer with the amphotropic retrovirus either, suggesting that the abundance of Pit2 is not the limitation.…”

“…The human Pit2 cDNA was a generous gift of Jean M Heard. 19 Enhanced GFP (Clontech, Palo Alto, CA, USA) was placed at the N terminus of the human Pit2 cDNA. This construct was then cloned into an adenovirus shuttle plasmid in which the expression of eGFP/human Pit2 fusion was driven by the CMV promoter.…”

A novel analytical method for in vivo phosphate tracking

“…Co-expression of a Na + /Pi transporter and the FRET sensor in COS-7 cells Similarly, COS-7 cells expressing the FLIPPi-30m sensor also did not reproducibly respond to perfused P i (data not shown). It is known that phosphate transport activities are down-regulated by external P i [28,29]. As an alternative means of increasing P i transport rates of the COS-7 cells, the human Na + /Pi cotransporter PiT2 was co-expressed along with the FLIPPi-30m sensor.…”