The Central Half of Pit2 Is Not Required for Its Function as a Retroviral Receptor

Abstract

The type III sodium-dependent phosphate (NaP i ) cotransporter, Pit2, is a receptor for amphotropic murine leukemia virus (A-MuLV) and 10A1 MuLV. In order to determine what is sufficient for Pit2 receptor function, a deletion mutant lacking about the middle half of the protein was made. The mutant supported entry for both viruses, unequivocally narrowing down the identification of the sequence that is sufficient to specify the receptor functions of Pit2 to its N-terminal 182 amino acids and C-terminal 170 amino acids.The type III sodium-dependent phosphate (NaP i ) symporter, Pit2, from humans is a receptor for amphotropic murine leukemia virus (A-MuLV) and the related strain 10A1 MuLV (13,22,23,25,40,44). The related Pit1 protein from humans (62% amino acid identity) is a receptor for gibbon ape leukemia virus, feline leukemia virus subgroup B, and 10A1 MuLV (22,24,39). The two human type III NaP i symporters show about 25% amino acid identity with the Neurospora crassa NaP i symporter 40). Several studies have aimed at identifying sequences that are critical for the viral receptor functions of Pit1 and Pit2. These studies used chimeras between Pit1 and Pit2 orthologs, Pit1 orthologs, human Pit1 and Pho-4, human Pit2 and Pho-4, and human Pit1 and Pit2 and Pho-4 as well as Pit1 and Pit2 mutants (3, 5-8, 12, 14-16, 22, 26-28, 31-33, 38, 42, 43). The results suggest that amino acids and/or sequences present in human Pit2 positions 67 to 91 (8), 107 to 141 (14), 517 to 530 (15), and 522 to 530 (28) or corresponding positions in related proteins (15,16,22,28,33) specify or are involved in A-MuLV receptor function. Moreover, human and mouse Pit1 chimera studies suggest that amino acids in the mouse and human Pit1 regions corresponding to human Pit2 sequence 522 to 530 also are involved in 10A1 receptor function (16); these results have been confirmed in studies of human Pit2 mutants (unpublished results).The design of the majority of the chimerical and mutant proteins (14-16, 22, 28, 33) was based on a membrane topology model for Pit1, Pit2, and Pho-4, which was suggested by Johann and coworkers on the basis of Kyte-Doolittle hydropathy plots (4,11,40) (Fig. 1B). Recently, new topology models that differ from the previous models have been proposed for Pit1 (7) and Pit2 (30) (Fig. 1A). If Pit1 and Pit2, however, exhibit different topologies in the membrane, the interpretation of which sequences are directly involved in receptor function for A-MuLV and 10A1 based on results obtained with Pit1/Pit2 chimeras may be incorrect. Moreover, one cannot in general exclude the possibility that amino acid exchanges in, for example, one part of Pit2 would lead to overall structural changes in another part of the protein, as shown for Pit1 by Farrell et al.(7). Results obtained with exchange mutants, therefore, may also potentially lead to incorrect interpretations concerning the identities of amino acids in Pit2 that are directly involved in A-MuLV and 10A1 receptor functions. It is obvious that these problems cannot be addresse...

Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

“…In another study, deletion of about the middle half of human Pit2 was found to reduce A-MLV and 10A1 entry by 75% to 90% in CHO K1 cells compared to entry via wild-type human Pit2 (5). In this Pit2 mutant, the caveolin-binding consensus sequence in positions 226 to 233 is deleted.…”

Phosphate transporters: a tale of two solute carrier families

“…Interestingly, the plant protein is lacking the large intracellular loop present in animal PiTs. Furthermore, Bottger and Pedersen (9) showed that PiT2 retains its retroviral receptor function after deletion of loop 7 as well as transmembrane domains 6 -7 (pink shading in Fig. 5).…”

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake

“…To this aim, we constructed hPiT1 and hPiT2 chimeric proteins expressing the eYFP acceptor or Rluc donor. Since structure-function studies have excluded a role of the large intracellular loop (iLoop) in Pi transport and retrovirus binding (59)(60)(61)(62), and showed no overlapping between iLoop and the highly hydrophobic domain (57), we substituted the iLoop with eYFP and Rluc sequences ( Figure 3C). When expressed in HEK293T, the chimeric hPiT1-eYFP or -Rluc and hPiT2-eYFP or -Rluc proteins could be visualized at the plasma membrane, as shown by confocal microscopy ( Figure 3D), enabling to study their role in detecting the variation of extracellular Pi levels.…”