INVITED REVIEWAn update on renal peptide transporters - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:36 |只看该作者 |倒序浏览 |打印

作者：HanneloreDaniel and IsabelRubio-Aliaga作者单位：Molecular Nutrition Unit, Technical University of Munich,D-85350 Freising-Weihenstephan, Germany $ c5 ~1 M) j' g- R! W* \

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      【摘要】
   The brush-border membraneof renal epithelial cells contains PEPT1 and PEPT2 proteins that arerheogenic carriers for short-chain peptides. The carrier proteinsdisplay a distinct surface expression pattern along the proximaltubule, suggesting that initially di- and tripeptides, either filteredor released by surface-bound hydrolases from larger oligopeptides, aretaken up by the low-affinity but high-capacity PEPT1 transporter andthen by PEPT2, which possesses a higher affinity but lower transportcapacity. Both carriers transport essentially all possible di- andtripeptides and numerous structurally related drugs. A unique featureof the mammalian peptide transporters is the capability ofproton-dependent electrogenic cotransport of all substrates, regardlessof their charge, that is achieved by variable coupling in protonmovement along with the substrate down the transmembrane potentialdifference. This review focuses on the postcloning research efforts tounderstand the molecular physiology of peptide transport processes inrenal tubules and summarizes available data on the underlying genes, protein structures, and transporter function as derived from studies inheterologous expression systems. ' j1 C- K# |  @( q; j3 U
      【关键词】 PEPT PEPT renal physiology localization functional analysis7 F6 O, ^2 D/ }' O0 `' k; g
   INTRODUCTION- \7 ~. u: B6 i" b

RENAL TUBULAR PEPTIDE TRANSPORT activity was originally discovered after intravenous infusion of theresistant dipeptide Gly-Sar in rats that resulted in a highaccumulation of the intact dipeptide in renal tissue ( 1, 3 ). Studies with renal brush-border membrane vesiclesestablished that renal apical peptide uptake was an electrogenic,proton-dependent uphill transport process for di- and tripeptides andrelated drugs mediated by two kinetically different systems (for areview, see Ref. 38 ). The underlying proteins, differingin transport characteristics, now designated as PEPT1 (SLC15A1) andPEPT2 (SLC15A2), have been identified, and the corresponding genes havebeen cloned from a variety of species ( 10, 24, 25, 39, 41, 52-54 ). The transporters have been expressed in variouscellular models, and information on structure, transport function, andregulation has been gathered by flux studies and electrophysiologicaltechniques. Expression analysis of transporter mRNA and protein by insitu hybridization and immunohistochemistry has extended our knowledgeof tissue distribution, particularly the renal zonation of PEPT1 andPEPT2 surface expression.

THE MOLECULAR ENTITIES OF APICAL PEPTIDE TRANSPORT) h; H5 V  T1 ]; y4 w& r# v
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PEPT1 and PEPT2 are polytopic integral membrane proteins with 12 predicted membrane-spanning domains and NH 2 - andCOOH-terminal ends facing the cytosol. Transporter architectureand membrane topology have not been studied systematically, andtherefore structural information is still mainly based on hydropathicanalysis of amino acid sequences. The mammalian PEPT1 proteins comprise701-710 amino acids, depending on the species ( 24, 25, 39, 53 ), and are highly glycosylated. As shown by in vitrotranslation studies in the presence of microsomes, PEPT1 has amolecular mass of ~75 kDa, whereas digestion with endoglycosidase Ashifted the mass to 63 kDa ( 53 ). The PEPT2 protein is alsoglycosylated, with a molecular mass of the mature protein of ~107 kDaand a nonglycosylated mass of 83 kDa ( 10 ). Westernblot analysis of protein preparations of intestine and kidneyidentified PEPT1 and PEPT2 immunoreactivity as glycosylated proteinswith molecular masses of ~75 and 100 kDa, respectively ( 47, 52 ). The Pept2 gene encodes a 729-amino acid protein( 10, 41, 52, 54 ) with 48% amino acid identity to PEPT1predominantly in transmembrane domains (TMD) and the lowest identity inthe large extracellular loop connecting TMD 9 and 10. The genomicorganization of the peptide transporter genes has also been elucidated.The murine Pept1 gene is estimated to be ~38 kb long,including a TATA-less promoter region ( 25 ), and bothmurine and human Pept1 genes possess 23 exons and 22 introns( 25, 70 ). The murine Pept2 gene is 34 kb long,consists of 22 exons and 21 introns ( 52 ), and also carriesa TATA-less promoter. The human Pept1 and Pept2 genes have been mapped to chromosomes 13q33-34 and 3q13.3-q21, respectively ( 39, 51 ). The murine Pept2 genewas localized in a syntenic region with human chromosome 3q13.3-q21, oncentral mouse chromosome 16 close to D16Mit4 and D16Mit59( 52 ).
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TISSUE DISTRIBUTION OF PEPT1 AND PEPT2 AND THEIR TUBULARLOCALIZATION

PEPT1 was first identified in the brush-border membrane of theepithelial cells of the small intestine and cloned from an intestinalcDNA library ( 24, 47 ). It was then also found in thebrush-border membrane of epithelial cells in the kidney proximal tubuleS1 segment ( 60 ). By immunostaining and transport analysis, PEPT1 has also recently been shown to be present in the apical membraneof bile duct epithelial cells ( 36 ).9 F" T- j5 D9 _1 P- V
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PEPT2 shows much broader expression and tissue distribution within theorganism. It was initially cloned from a renal cDNA library anddetected in brush-border membranes of cells in S2 and S3 segments( 10, 60 ). More recently, PEPT2 has been identified in theperipheral nervous system, in the membranes and cytoplasm of satelliteglial cells surrounding the ganglionic neurons ( 29 ), andin the central nervous system. In situ hybridization analysis revealedits presence in the brain in astrocytes, ependymal and subependymalcells, and in epithelial cells of the choroid plexus ( 8 ).In the lung, PEPT2 is expressed in type II pneumocytes and in theapical membrane of tracheal and bronchial epithelial cells( 31 ). Furthermore, a recent study has shown thelocalization of PEPT2 in the apical membranes of the epithelial cellsof the terminal duct and glandules as well as the main and segmental ducts in the lactating mammary gland ( 30 ). By RT-PCR,expression of PEPT2 mRNA was also demonstrated in the spleen, colon,and pancreas ( 20 ).2 M0 M, F) e& E8 H
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Along the nephron, peptide transporters are only found in the proximaltubule (see Fig. 1 ), both by analysis offunction as well as by immunolocalization. PEPT1 has been detected inthe brush border of the epithelial cells of the convoluted proximal tubule, in the S1 segment, with progressively weaker expression indeeper cortical regions ( 60 ). PEPT2 is localized in thebrush-border membrane of S2 and S3 segment cells of the proximaltubule, with strong immunostaining in the outer stripe but not theinner stripe of the outer medulla, including the medullary raysprotruding into the deeper cortical regions ( 60 ). A recentstudy employing renal brush-border membrane vesicles has functionallyverified this distribution of the peptide transporters in the proximal tubule ( 40 ). Whereas in membranes prepared from outercortex cells, two systems mediated the transport of the model dipeptide Gly-Sar, in membranes from cells of the outer medulla only one systemwas kinetically detectable. Biochemical studies in the 1980s withbrush-border membrane vesicles already showed the presence of alow-affinity, high-capacity and a high-affinity, low-capacity transportsystem ( 18 ). Studies in renal cell lines such asMadin-Darby canine kidney cells that possess PEPT1 activity andLLC-PK 1 and SKPT cells that express PEPT2 activity andvarious heterologous expression systems have established that PEPT1 isthe low-affinity-type transporter. PEPT2 shows under the sameexperimental conditions much higher affinities for almost all testedsubstrates. The different kinetic properties of PEPT1 and PEPT2 areshown with -aminolevulinic acid as a substrate in Fig. 1 A. The maximal transport capacity of PEPT1 appears to be5-15 times higher than that of PEPT2 for most substrates and inall cellular systems analyzed so far. This supports the contention thatpeptides are handled sequentially in the kidney, first by thelow-affinity, high-capacity transporter PEPT1 and then by thehigh-affinity, low-capacity transporter PEPT2.
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Fig. 1. Renal peptide transporters PEPT1 and PEPT2. PEPT1 and PEPT2 arelocalized in the apical membrane of epithelial cells of the proximaltubule with a distinct expression pattern. A : comparativetransport rates for the substrate -aminolevulinic acid by PEPT1 orPEPT2 under identical experimental conditions, which demonstrate thatPEPT1 is the low-capacity, high-affinity transport system and PEPT2 thehigh-affinity, low-capacity carrier. B : different affinitiesof the 2 carriers for a large set of identical Ala-Xaa (filled symbols)and Xaa-Ala (grey symbols) dipeptides. A, D, E, F, G, H, K, L, P, R, S,W, and Y: single amino acids; A, -aminobutyric acid; Ai,aminoisobutyric acid; Nl, norleucine; Pi, pipecolic acid; Sa,sarcosine. C : simplified model in the role of renal peptidetransporters in metabolism of proteins, di- and tripeptides, and aminoacids in renal tubular cells. p, Proteins; AT, amino acidtransporter.
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That the transport activity of the renal peptide transporters can alsobe visualized is shown in Fig. 2 by useof the fluorescent dipeptide conjugate D -Ala-Lys- N -7-amino-4-methylcoumarin-3-acetic acid ( D -Ala-Lys- AMCA), a known substrate of PEPT2 andPEPT1 ( 28, 31 ). Rat kidney perfused with D -Ala-Lys-AMCA reveals strong fluorescent staining only inepithelial cells of the proximal tubule, and staining is almostcompletely blocked by the dipeptide Gly-Gln when coadministered.( ?+ E5 p' C3 V$ \" V
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Fig. 2. Visualization of peptide transport in rat kidney by D -Ala-Lys-AMCA. Uptake experiments were carried out byadministration of 25 µM fluorescent dipeptide derivative D -Ala-Lys- N -7-amino-4-methylcoumarin-3-aceticacid ( D -Ala-Lys-AMCA) into the dorsal caudal vein of rats. Inset : for inhibition studies 25 µM D -Ala-Lys-AMCA and 1 mM unlabeled Gly- L -Glnwere used. After 30 min, a 10-min kidney perfusion with ice-coldunlabeled minimal essential media was performed, followed by perfusionfixation with freshly prepared 4% paraformaldehyde (PFA) in PBS at pH7.4 for 5 min. Kidneys were subsequently immersed in a sucrose-PBSsolution adjusted to 800 mosmol/kgH 2 O for cryoprotectionand shock-frozen in liquid nitrogen-cooled isopentane. After beingprocessed into 10-µm cryostat sections, the slides were viewed usingepifluorescence detection.& ^0 u; N( o* K4 h
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THE SUBSTRATE SPECIFICITY OF PEPT1 AND PEPT2
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One of the striking features of the mammalian peptide transportersis their apparent promiscuity, because both carriers can transportessentially every possible di- and tripeptide. This means that thereare at least 400 different dipeptides and 8,000 different tripeptidesthat could serve as substrates, and these peptides vary considerably inmolecular size, charge, and polarity. Transport occurs in all casesenantioselectively, but di- and tripeptides containing a D -amino acid, particularly when placed in theNH 2 terminus, are also accepted as substrates, although with lower affinity ( 19 ). This further increases thenumber and variety of potential substrates. Moreover, there are drugs such as numerous aminocephalosporins, selected angiotensin-converting enzyme inhibitors, peptidase inhibitors, and a variety of novel prodrugs that are also taken up by both carriers (for a review, seeRefs. 34 and 71 ). Another puzzling and still not fully explained characteristic of the transporters is that, regardless of thesubstrates' net charge at a given pH, transport occurs electrogenically and is always associated with the cotransport ofprotons, as shown by simultaneous recordings of intracellular pHchanges. Electrogenicity in the transport of charged substrates ispresently best explained by a variable flux coupling ratio forsubstrate-to-proton cotransport ( 62 ). Although there are some differences in the affinities of the different substrate groups,PEPT1 and PEPT2 essentially can transport all di- and tripeptides in anelectrogenic mode, and this is important in view of their role inreabsorption and the most efficient conservation of peptide-bound amino acids.7 t0 I& e; x, J1 B

To unravel the secret of the broad substrate spectrum accepted by PEPT1and PEPT2, numerous studies have been conducted. Large sets ofdipeptides, amino acid derivatives, or peptidomimetics have been probedfor transport in competition experiments utilizing cells expressingeither one of the transporters, by the measurement ofsubstrate-mediated transport currents in Xenopus laevis oocytes expressing PEPT1 or PEPT2, or by competition experiments in the yeast Pichia pastoris heterologously expressing either oneof the mammalian transporters ( 11, 12, 14, 21, 22, 66, 67 ). One important finding for understanding the"multispecificity" of the peptide transporters was that neitherPEPT1 nor PEPT2 requires a peptide bond in a substrate and that theminimal structural requirement in a substrate for binding and transportis a simple carbon chain that separates the oppositely chargedNH 2 and COOH 500 ( 21 ). In thecase of PEPT2, we recently showed by the rational design of a large setof test compounds that the much higher affinity of PEPT2 for the samesubstrates is based on the requirement of an additional carbonylfunction, but not COOH, within the backbone of a substrate( 67 ). These minimal substrate requirements are perfectlymatched by -aminolevulinic acid (ALA), which was shown to be a goodsubstrate of PEPT2 and PEPT1 ( 20 ). This compound serves asa precursor of phorphyrin synthesis and is used in the treatment ofvarious cancers by photodynamic therapy, whereby ALA is thenadministered either exogenously or topically. ALA is known to possesspronounced renal toxicity by selective accumulation in tubular cells( 35 ), to which PEPT1 and PEPT2 might contribute byeffective reabsorption of ALA from tubular fluids.1 z' ]- V) ^- J

In the case of normal di- and tripeptides, the substrate binding sitein both carriers can accept all the various side chains of amino acids,but it appears that the side chains are accommodated in asymmetricbinding pockets. This has also been demonstrated by the selectivity ofnewly identified PEPT1 and PEPT2 inhibitors with [Z(NO 2 )]groups attached to the -amino group in Lys dipeptides ( 37, 68 ). Only when present in a particular position within thedipeptide, the Lys-[Z(NO 2 )] derivatives served asinhibitors; otherwise, they were substrates. The bulkiness and/orcharge of the amino acid side chains alter the affinity of dipeptidesfor interaction with PEPT1 and PEPT2. This is shown in Fig. 1 B, in which the same substrates have been used to determinethe apparent K i values for inhibition of D -Phe-Ala transport mediated by PEPT1 or PEPT2 underidentical experimental conditions in transgenic P. pastori scells expressing either rabbit PEPT1 or PEPT2. It should be emphasizedhere that affinities for the same substrate determined in differentexpression systems can vary considerably, and this is most likely dueto marked effects of membrane potential and pH on substrate bindingaffinity. All dipeptides tested have generally higher affinities forPEPT2 than for PEPT1, but the differences can be very modest, as in thecase of Gly-Asp and Lys-Glu, or ~20-fold, as in the case of Lys-Gly.
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Although there are also differences in the affinities ofpeptidomimetics, the same classes of xenobiotics such as the various aminocephalosporins and selected angiontensin-converting enzyme inhibitors are taken up by both carriers. A recently proposed templatefor binding of a substrate to PEPT1 explains most of the structural andconformational requirements known thus far for the interaction ofsubstrates with the substrate binding domain and possibly also forPEPT2 with similar but not identical structural substrate requirements( 6 ).
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INTEGRATING THE PEPTIDE TRANSPORTERS INTO OVERALL AMINO ACIDHOMEOSTASIS

Studies of the plasma clearance of dipeptides indicate that therenal tubule is the only tissue capable of accumulating short-chain peptides in concentrations that are greater than their plasma concentrations (for a review, see Refs. 2 and 17 ). Thisobservation is also in accordance with the recent findings on thelocalization of the peptide transporters PEPT1 and PEPT2 in extrarenaltissues. Although PEPT1 is found in the intestine and in bile ductepithelium ( 36, 47 ), in these locations it does notcontribute to the clearance of di- and tripeptides from thecirculation. PEPT2 has been localized to the apical membrane oftracheal and bronchial epithelial cells in the lung ( 31 )and ductal cells of the lactating mammary gland ( 30 ), butby this localization PEPT2 cannot also play a role in the clearance ofshort-chain peptides from the blood. Functional studies using isolatedchoroid plexus suggest that PEPT2 there is located in the apicalmembrane, where it may mediate the removal of di- and tripeptides fromthe cerebrospinal fluid to the blood ( 46 ) rather thenserving as an import system.

In the kidney, the peptide transporters contribute to the homeostasisof amino acids in the organism along with several classes of amino acidtransporters located as well in the apical membrane of tubular cells(for a review, see Ref. 48 ). Di- and tripeptides aredelivered to PEPT1 and PEPT2 in epithelial cells either by surfacehydrolysis of larger oligopeptides or by glomerular filtration (seeFig. 1 ). Data from animal studies suggest that up to 50% ofcirculating plasma amino acids might be peptide bound and of those25-50% could be di- and tripeptides ( 27, 57, 58 ). However, the composition of this di-/tripeptide fraction circulating inplasma or provided in the filtrate is not known. Moreover, only a fewindividual dipeptides have been analyzed in plasma. The highestconcentration of a known individual dipeptide (Cys-Gly) in plasma and,based on its free filtration also in tubular fluids, 50 49 ). Cys-Gly, which is a breakdown product ofglutathionine, is obviously cleared quite well in the kidney, with meanconcentrations in human urine samples of 7.4 µM. In a concentrationrange of 50 µM, both peptide transporters may contribute to thereabsorption of Cys-Gly. However, the regional distribution of thepeptide transporter along the nephron and recent functional studiessuggest that the di- and tripeptides are handled sequentially in thekidney, first by the low-affinity, high-capacity system PEPT1, and thenby the high-affinity, low-capacity system, which would dominate atlower substrate concentrations ( 40 ). The renal zonationand the different affinities and transport capacities of the twotransporters in concert allow maximal reabsorption capacity and highestefficiency for conservation of peptide-bound amino acids and thereduction of renal losses of amino acid nitrogen.& u8 l$ D+ {5 S3 z) u

Whereas the apical uptake of di- and tripeptides has been studiedextensively and may represent the main route for renal peptide clearance from circulation, recent reports have suggested that abasolateral peptide transporter may also contribute to the selective uptake of peptides into the kidney (see Fig. 1 ). Studies in Madin-Darby canine kidney cells, which display characteristics of cells of distaltubules or collecting ducts, and LLC-PK 1 cells, whichdisplay the characteristics of cells of the proximal tubule, express a transport activity for influx across the basolateral membrane that isdistinctly different from the peptide transporter type on the apicalside ( 56, 64 ). These studies suggest that another peptidetransporter has to be found and cloned. However, studies in intactperfused rat kidney in vitro have failed to detect any peritubulardipeptide (Gly-Sar) uptake when glomerular filtration was prevented( 43 ). The basolateral membrane therefore remains as thedark site of epithelial peptide transport.
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When peptides are transported into the cell from the apical side, mostof them will be cleaved rapidly by cytosolic peptidases because thekidney exhibits the highest intracellular hydrolase activity againstshort-chain peptides ( 2 ) (see Fig. 1 C ).Dipeptides resistant to hydrolysis or peptidomimetics that cannot becleaved (i.e., aminocephalosporins) may even be released intact backinto the circulation across the basolateral membrane ( 7 ).Whether this efflux is mediated by an as yet unknown peptidetransporter or one of the organic anion and or cation transporters ofthe OCT and OAT series, which can also transport zwitterioniccompounds, remains to be determined. Immunodetection studies withPEPT1- or PEPT2-specific antisera have yet not shown anycross-reactivity with proteins in renal basolateral membranes. Someconflicting results have been obtained for a immunoreactive protein inrenal lysosomes. A pH-dependent dipeptide transport activity, similar to PEPT1 but with a lower affinity, has been identified in renal lysosomes, and this system may be responsible for the export of di- andtripeptides from the lysosomes to the cytoplasma( 75 ). In Western blot analysis of lysosomalproteins, a PEPT1 antiserum recognized a specific band with a mass of~78 kDa. The lysosomal peptide transporter could contribute to renalclearance of short-chain peptides, and the cellular delivery of freeamino acids for protein synthesis and metabolism can be anticipated asmost of the proteins filtered in the glomerulus are reabsorbed in therenal proximal tubule by endocytosis mediated by binding to endocyticreceptor proteins such as megalin or cubilin ( 16 ) (seeFig. 1 ). After endocytosis, the proteins accumulate in lysosomes fordegradation and the lysosomal peptide transporter could provide a routefor the transfer of released di- and tripeptide from a region of low dipeptidase activity (lysosomes) to a region of high dipeptidase activity (cytosol). This compartmentation could also prevent a premature osmotically induced swelling and rupture of lysosomes ( 75 ). The question of whether the lysosomal peptidetransporter is PEPT1 or one of the PHT histidine/peptide transportersrecently cloned, which are found in lysosomes when expressedheterologously ( 33, 55, 74 ), or a completely newtransporter remains to be determined.' ?) f6 @2 V1 w1 p

REGULATION OF EXPRESSION LEVEL AND TRANSPORT ACTIVITY OF PEPT1 ANDPEPT2
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Most of the studies on regulation of peptide transport have beenperformed with PEPT1 in view of its intestinal role in the transport ofdietary peptides and drugs. Comparative analysis using Northern andWestern blotting techniques has shown an increase in the expression ofPEPT1 in the small intestine immediately after birth and expression inthe colon, which disappeared in adult stage ( 59 ). In thekidney, the expression levels of PEPT1 and PEPT2 increased steadilyover time to reach maximal levels 2 wk after birth. A differentialadaptative response of PEPT1 and PEPT2 has recently been shown in amodel of chronic renal failure. The 5 6 nephrectomized rat iswidely used as a model for the study of the progression of renal damage resulting from the reduction of nephron mass. In this model, a pronounced upregulation of PEPT2 mRNA and protein expression 2 wk afterrenal ablation occurred, whereas mRNA and protein levels of PEPT1 didnot change ( 63 ). This is the first study to show theregulation of the peptide transporter PEPT2 in kidney in vivo, and thefindings may also be of importance for the pharmacokinetics of thedrugs transported by PEPT2 in patients suffering from chronic renal failure.

Dietary regulation of the intestinal peptide transporter PEPT1 has beendemonstrated in several studies ( 23, 61, 72 ). An increasein the protein content of a diet increased the uptake of a modeldipeptide in the small intestine of rats as well as the mRNA andproteins levels of PEPT1 ( 23, 61 ). Moreover, addition ofselected amino acids or dipeptides to media in cell culture increaseddipeptide uptake, and in most cases also the mRNA and proteins levelsof PEPT1 ( 61, 72 ). This stimulation of gene transcriptionmay be a consequence of an activation of the Pept1 promotervia selected amino acids and dipeptides ( 61 ). Promoteranalysis studies employing a luciferase reporter assay identified aregion that responded to all dipeptides tested and also to selectedamino acids, such as Lys, Arg, and especially Phe. This regioncomprises 254 bp and contains an amino acid response-like element asfound in the asparagine synthetase gene ( 32 ).Rat and mouse Pept1 and the murine Pept2 promoterregions contain these amino acid response-like elements with a bpsubstitution in the fifth bp ( 25, 52 ). Whether these cis elements play a role in controlling gene expression inthe kidney in response to cellular availability of free amino acids anddipeptides needs to be determined. However, studies in humans suggestthat starvation reduces renal peptide transport activity, asdemonstrated by a reduction in removal of the intravenously infuseddipeptide Gly-Gln by the kidney ( 4, 42 ). In the yeast Saccharomyces cerevisiae, gene expression of Ptr-2, the yeast peptide transporter ( 50 ), isupregulated at the expression level by selected dipeptides thatactivate an ubiquitin-dependent proteolytic pathway ( 69 ). Whether such a mechanism controls the expression levels of mammalian peptide transporters also has to be investigated.! _& H2 i3 I& ]& L6 X0 [7 u. e
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Receptor ligand-induced acute regulation of PEPT1 and PEPT2 transportactivities has so far only been studied in cell lines expressing PEPT1,such as intestinal Caco-2 and renal Madin-Darby canine kidney cells, orin renal LLC-PK 1 and SKPT cells expressing PEPT2. In Caco-2cells, insulin ( 65 ), leptin ( 15 ), 1 - and 2 -adrenergic receptor ligands suchas ( )pentazocine ( 26 ), and clonidine ( 9 ) allwere shown to stimulate uptake of dipeptides or -lactams via PEPT1.On the other hand, either exposure to two immunosuppressive agents,tacrolimus and cyclosporin A ( 44 ), or exposure to thyroidhormone ( 5 ) or long-term basolateral stimulation with EGFinhibited the uptake of dipeptides via PEPT1 in Caco-2 cells( 44 ). Moreover, it has been demonstrated that activationof signaling pathways that involve protein kinases C and A changes thekinetic properties of PEPT1 and PEPT2 in intestinal and renal celllines ( 13, 73 ). Whether these in vitro observations are ofphysiological importance in vivo in renal tissues remains to be determined.
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CONCLUDING REMARKS' {# K$ r! j) V

When expression cloning of the mammalian peptide transportersallowed one to relate the transport phenomena observed in intact tissues or tissue preparations to distinct proteins, exciting newfindings on the molecular physiology of these unique proton-coupled transporters have been obtained. Heterologous expression studies ofcloned transporters yielded a wealth of information on substrate specificity, coupling stoichiometry, and translocation mechanisms, andbiophysical analyses of transporter mutants and chimeras provide firstclues on the domains within the transporter proteins that contribute tosubstrate binding and determine specificity. New data on tissuedistribution, particularly with respect to PEPT2, have been gatheredthat are still puzzling in view of the physiological role of thetransporter in these tissues. We are now awaiting information on themetabolic consequences of the lack of renal peptide transporters inexperimental animals from targeted gene inactivation.' h8 g7 n* w3 Y# M/ j+ }
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10. Boll, M,Herget M,Wagener M,Weber WM,Markovich D,Biber J,Clauss W,Murer H,andDaniel H. Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter. Proc Natl Acad Sci USA 93:284-289,1996 .
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11. Borner, V,Fei YJ,Hartrodt B,Gaapathy V,Leibach FH,Neubert K,andBrandsch M. Transport of amino acid aryl amides by the intestinal H /peptide cotransport system, PEPT1. Eur J Biochem 255:698-702,1998  .) F: B4 y' N9 Z+ \9 Q! ^
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12. Brandsch, M,Knutter II,Thunecke F,Hartrodt B,Born II,Borner VV,Hirche F,Fischer G,andNeubert K. Decisive structural determinants for the interaction of proline derivatives with the intestinal H /peptide symporter. Eur J Biochem 266:502-508,1999  ., a* b6 a* M, j  |3 o7 j, n

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14. Brandsch, M,Thunecke F,Kullertz G,Schutkowski M,Fischer G,andNeubert K. Evidence for the absolute conformational specificity of the intestinal H /peptide symporter, PEPT1. J Biol Chem 273:3861-3864,1998 .

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