Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms.
l. Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport. PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes. In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity. In addition, both transporters show differences in pH dependence and substrate specificity. 2. As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1. Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique. 3. Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters. We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region.
Döring
F
F
Institute of Nutritional Sciences, University of Giessen, Germany.
Dorn
D
D
Bachfischer
U
U
Amasheh
S
S
Herget
M
M
Daniel
H
H
D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002434:Cefadroxil / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli; D005071:Evoked Potentials; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009865:Oocytes / Q000378:metabolism; D000074011:Peptide Transporter 1; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000502:physiology*; D013379:Substrate Specificity; D027981:Symporters; D014981:Xenopus
D002352:Carrier Proteins; D004151:Dipeptides; D000074011:Peptide Transporter 1; D011993:Recombinant Fusion Proteins; C082186:SLC15A1 protein, human; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; D002434:Cefadroxil; C039552:phenylalanylalanine