Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms.

Abstract

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.

Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro586

“…To date, some amino acid residues or domains in PEPT1 have been revealed functionally important, and the results in the present study are largely compatible with those earlier findings: 1) Previous studies on PEPT1 and PEPT2 chimeras (Doring et al, 1996) suggested that the large extracellular loop between TM9 and TM10 is not essential for phenotypical characteristics of transporter function. This observation is in good agreement with our results: five cSNPs located on this loop have little influence on transporter function in terms of pH dependence, substrate uptake kinetics, and inhibition specificity.…”

“…In addition to identification of those functionally important residues in PEPT1, it has been proposed that the N-terminal one-half including TM7, TM8, and TM9 is the region of PEPT1 responsible for proton and substrate recognition (Doring et al, 1996;Fei et al, 1998;Terada et al, 2000), and a recent study with cysteine mutagenesis on PEPT1 revealed that TM5 may be part of the substrate translocation pathway (Kulkarni et al, 2003). Additionally, it has been speculated that the C-terminal region, although not directly involved in substrate recognition and binding, may be important for either protein trafficking or functional regulation (Doring et al, 1996). Located on the membrane boundary of TM10 in C-terminal portion of PEPT1, Pro 586 was shown not to be important for protein function in terms of pH-dependent uptake, Gly-Sar affinity, and inhibitor specificity, because P586L behaved similarly to the reference gene with respect to these parameters.…”

Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish ( Chionodraco hamatus ) PEPT1 transporter

“…This region is poorly conserved among vertebrate PEPT1 orthologs. Moreover, this region is not involved in the transport activity, as assessed by computational, site-directed mutagenesis, chimeric, and other approaches (36)(37)(38)(39)(40)(41)(42)(43). Insertion of the VDMSRKS domain into this region does contribute to the cold adaptation of the protein.…”

Tissue distribution of a peptide transporter mRNA in sheep, dairy cows, pigs, and chickens.

“…More recently, Doring et al (1997) performed a functional analysis of peptide transport by using a chimeric transporter obtained from the intestinal and renal isoforms.…”