N-terminal halves of rat H⁺/peptide transporters are responsible for their substrate recognition

Purpose. Peptide transporters PEPT1 and PEPT2 differ substantially in their substrate affinity and recognition. The aim of this study is to define the structural domains which influence the functional characteristics of both transporters. Methods. Two kinds of chimeric peptide transporters (PEPT-N1C2 and PEPT-N2C1) were constructed, and their functional characteristics were compared with those of wild-type transporters in stable transfectants. Results. PEPT-N1C2, the N-terminal half of rat PEPT1 and the C-terminal half of rat PEPT2, and the reciprocal chimera PEPT-N2C1 were functionally expressed in LLC-PK₁ cells. The pH-profiles of [¹⁴C] glycylsarcosine uptake by PEPT-N1C2 and PEPT-N2C1 were close to those of PEPT1 and PEPT2, respectively. Substrate recognition for PEPT-N1C2 and PEPT-N2C1 was also similar to that of PEPT1 and PEPT2, respectively. However, substrate affinities for PEPT-N1C2 were higher than those for PEPT1, although those for PEPT-N2C1 and PEPT2 were comparable. Conclusions. These results indicate that functional regions which are associated with the extracellular pH changes and are responsible for substrate recognition of PEPT1 and PEPT2 may be located in the N-terminal halves of the proteins. In addition, it is suggested that the domain to affect the substrate affinity exists in the C-terminal as well as in the N-terminal half of rat PEPT2.