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M A Hediger

Publications and source records attributed to M A Hediger.

At least 109 records · Page 6Linked to original sources
PubMed1993

Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid.

Maintenance of a stable internal environment within complex organisms requires specialized cells that sense changes in the extracellular concentration of specific ions (such as Ca2+). Although the molecular nature of such ion sensors is unknown, parathyroid cells possess a cell surface Ca(2+)-sensing mechanism that also recognizes trivalent and polyvalent cations (such as neomycin) and couples by changes in phosphoinositide turnover and cytosolic Ca2+ to regulation of parathyroid hormone secretion. The latter restores normocalcaemia by acting on kidney and bone. We now report the cloning of complementary DNA encoding an extracellular Ca(2+)-sensing receptor from bovine parathyroid with pharmacological and functional properties nearly identical to those of the native receptor. The novel approximately 120K receptor shares limited similarity with the metabotropic glutamate receptors and features a large extracellular domain, containing clusters of acidic amino-acid residues possibly involved in calcium binding, coupled to a seven-membrane-spanning domain like those in the G-protein-coupled receptor superfamily.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the vasopressin-regulated urea transporter.

Urea is the principal end product of nitrogen metabolism in mammals. Movement of urea across cell membranes was originally thought to occur by lipid-phase permeation, but recent studies have revealed the existence of specialized transporters with a low affinity for urea (Km > 200 mM)2. Here we report the isolation of a complementary DNA from rabbit renal medulla that encodes a 397-amino-acid membrane glycoprotein, UT2, with the functional characteristics of the vasopressin-sensitive urea transporter previously described in in vitro-perfused inner medullary collecting ducts. UT2 is not homologous to any known protein and displays a unique pattern of hydrophobicity. Because of the central role of this transporter in fluid balance and nitrogen metabolism, the study of this protein will provide important insights into the urinary concentrating mechanism and nitrogen balance.

Amino Acid Sequence↗
PubMed1993

Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.

Electroneutral Na+:Cl- cotransport systems are involved in a number of important physiological processes including salt absorption and secretion by epithelia and cell volume regulation. One group of Na+:Cl- cotransporters is specifically inhibited by the benzothiadiazine (thiazide) class of diuretic agents and can be distinguished from Na+:K+:2Cl- cotransporters based on a lack of K+ requirement and insensitivity to sulfamoylbenzoic acid diruetics like bumetanide. We report here the isolation of a cDNA encoding a thiazide-sensitive, electroneutral sodium-chloride cotransporter from the winter flounder urinary bladder using an expression cloning strategy. The pharmacological and kinetic characteristics of the cloned cotransporter are consistent with the properties of native thiazide-sensitive sodium-chloride cotransporters in teleost urinary bladder and mammalian renal distal tubule epithelia. The nucleotide sequence predicts a protein of 1023 amino acids (112 kDa) with 12 putative membrane-spanning regions, which is not related to other previously cloned sodium or chloride transporters. Northern hybridization shows two different gene products: a 3.7-kb mRNA localized only to the urinary bladder and a 3.0-kb mRNA present in several non-bladder/kidney tissues.

Amino Acid Sequence↗
PubMed1993

Localization of the Na+/glucose cotransporter gene SGLT2 to human chromosome 16 close to the centromere.

The chromosomal location of the gene SGLT2, which is presumed to encode a low-affinity Na+/glucose cotransporter, has been determined using a panel of rodent-human somatic cell hybrids. Southern blot analysis of genomic DNA from 16 different hybrids shows that SGLT2 is located on chromosome 16. Analysis of three additional hybrids that selectively retain all or part of human chromosome 16 demonstrates that SGLT2 is located close to the centromere in band p11.2 of the chromosome.

Animals↗
PubMed1993

The elusive transporters with a high affinity for glutamate.

Removal of glutamate from the synaptic cleft is an essential component of the transmission process at glutamatergic synapses. This requirement is fulfilled by transporters that have a high affinity for glutamate and exhibit a unique coupling to Na+, K+ and OH- ions. Independently, three groups have succeeded in cloning cDNAs encoding high-affinity Na(+)-dependent glutamate transporters. These transporters are structurally distinct from previously characterized neurotransmitter transporters and show sequence identity with prokaryotic glutamate and dicarboxylate transporters. In addition, they exhibit significant differences in their structure, function and tissue distribution. This review compares and contrasts these differences, and incorporates into the existing body of knowledge these new breakthroughs.

Amino Acid Transport System X-AG↗
PubMed1993

A new family of neurotransmitter transporters: the high-affinity glutamate transporters.

An essential component of the transmission process at glutamatergic synapses is the removal of glutamate from the synaptic cleft. This is achieved by powerful transport systems which have a high affinity for glutamate and exhibit a novel coupling to inorganic ions. Transporters situated on presynaptic termini sequester glutamate directly from the synaptic cleft. In concert, transporters situated on glial cells maintain a low extracellular glutamate concentration, thereby establishing a diffusion gradient favoring movement of glutamate out of the synaptic cleft. Maintenance of a low extracellular glutamate concentration also serves to protect neurons from the excitotoxic action of glutamate. Despite the physiological importance of the glutamate transporters, little information has been available on their molecular structures. This gap, however, has begun to be bridged with the recent cloning of three species of eukaryotic glutamate transporters. The purpose of this review is to summarize the results of these three cloning successes, to compare and contrast the three novel transporters, and to reinterpret, in the light of these recent breakthroughs, information from previous studies.

Amino Acid Sequence↗
PubMed1993

Cloning and chromosomal localization of a human kidney cDNA involved in cystine, dibasic, and neutral amino acid transport.

We have recently cloned, sequenced, and characterized a rat kidney cDNA (D2) that stimulates cystine as well as dibasic and neutral amino acid transport. In order to evaluate the role of this protein in human inherited diseases such as cystinuria, we have isolated a human D2 clone (D2H) by low stringency screening of a human kidney cDNA library using the radiolabeled D2 insert as a probe. The D2H cDNA is 2284 nucleotides long and encodes a 663 amino acid protein that is 80% identical to the rat D2 amino acid sequence and 86% to that of the rabbit homologue rBAT. Microinjection of in vitro transcribed D2H cRNA into Xenopus oocytes induced uptake of cystine as well as dibasic and neutral amino acids in a pattern similar to that of rat D2 and rabbit rBAT. Both neutral and dibasic amino acids inhibited the D2H-induced uptake of cystine. Northern blot analysis demonstrated that D2H, like D2 and rBAT, is expressed strongly in the kidney and intestine. Southern blot analysis of genomic DNA from a panel of mouse-human somatic cell hybrids showed that the human gene for D2H resides on chromosome 2.

Amino Acid Sequence↗
PubMed1993

The type I iodothyronine 5'-deiodinase messenger ribonucleic acid is localized to the S3 segment of the rat kidney proximal tubule.

A complementary DNA clone encoding the type I iodothyronine 5'-deiodinase (5'DI), which converts T4 to T3, has been isolated recently. The 5'DI messenger RNA (mRNA) is most abundant in kidney and liver. To gain insight into the function of 5'DI in the kidney, Northern blot analysis was used to localize the expression of this mRNA. Our results show that 5'DI mRNA was expressed in the cortex and outer medulla, but not inner medulla and papilla, indicating that there are regional differences in its expression. Using in situ hybridization, we demonstrate that the 5'DI hybridization signal is localized predominantly over tubules in the outer stripe of the outer medulla and in medullary rays, suggesting localization to proximal tubules. To identify which tubular cells express 5'DI mRNA, we compared the profile of 5'DI message from in situ hybridization with the immunostaining of adjacent sections with proximal tubular S1, S2, and S3 segment-specific antibodies. Most of the 5'DI antisense complementary RNA hybridized to the same proximal tubular cells with which the S3-specific antibody, anti-ecto-ATPase, reacted. Cells staining with an S1 or S2 segment antibody showed little, if any, 5'DI mRNA. We conclude that the expression of 5'DI mRNA is restricted to the tubular cells of the proximal S3 segment. The S3 segment is also known to express high levels of proteins required for glutathione synthesis consistent with the requirement for a reduced thiol cofactor for iodothyronine deiodination by the 5'DI pathway.

Animals↗
PubMed1992

Primary structure and functional characterization of a high-affinity glutamate transporter.

Glutamate transport across plasma membranes of neurons, glial cells and epithelial cells of the small intestine and kidney proceeds by high- and low-affinity transport systems. High-affinity (Km 2-50 microM) transport systems have been described that are dependent on Na+ but not Cl- ions and have a preference for L-glutamate and D- and L-aspartate. In neurons high-affinity glutamate transporters are essential for terminating the postsynaptic action of glutamate by rapidly removing released glutamate from the synaptic cleft. We have isolated a complementary DNA encoding an electrogenic Na(+)- but not Cl(-)-dependent high-affinity glutamate transporter (named EAAC1) from rabbit small intestine by expression in Xenopus oocytes. We find EAAC1 transcripts in specific neuronal structures in the central nervous system as well as in the small intestine, kidney, liver and heart. The function and pharmacology of the expressed protein are characteristic of the high-affinity glutamate transporter already identified in neuronal tissues. The abnormal glutamate transport that is associated with certain neurodegenerative diseases and which occurs during ischaemia and anoxia could be due to abnormalities in the function of this protein.

Amino Acid Sequence↗
PubMed1992

The 4F2 antigen heavy chain induces uptake of neutral and dibasic amino acids in Xenopus oocytes.

The 4F2 cell surface antigen is a disulfide-linked heterodimer induced during the process of cellular activation and expressed widely in mammalian tissues (Parmacek, M. S., Karpinski, B. A., Gottesdiener, K. M., Thompson, C. B., and Leiden, J. M. (1989) Nucleic Acids Res. 17, 1915-1931). The human heavy chain component, a type II membrane glycoprotein, has 29% identity to the amino acid transport-related protein encoded by the recently cloned rat D2 cDNA. We have demonstrated that Xenopus oocytes injected with in vitro transcribed cRNA from D2 take up cystine and dibasic and neutral amino acids (Wells, R. G., and Hediger, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600). In the present study, we examine the role of the human 4F2 heavy chain in amino acid transport. In vitro transcribed 4F2 cRNA was injected into Xenopus oocytes which were assayed for the uptake of radiolabeled amino acids. Our results show that cRNA from 4F2 stimulates the uptake of dibasic and neutral amino acids into oocytes at levels up to 3-fold higher than for water-injected control oocytes. There is no demonstrable uptake of cystine. Uptake is saturable, with characteristics of high affinity transport, and inhibition data suggest that uptake occurs via a single transporter. Dibasic amino acids are taken up by both 4F2 and D2 cRNA-injected oocytes in a sodium-independent manner. In contrast, 4F2-induced but not D2-induced neutral amino acid uptake has a significant component of sodium dependence. Likewise, neutral amino acids in excess inhibit the 4F2-induced uptake of radiolabeled arginine but not leucine in a sodium-dependent manner. The 4F2-induced uptake we observe most likely represents the activity of a single transport system with some characteristics of systems y+, b0,+, and B0,+. We suggest that 4F2 and D2 represent a new family of proteins which induce amino acid transport with distinct characteristics, possibly functioning as transport activators or regulators.

Amino Acids↗
PubMed1992

Cloning of a rat kidney cDNA that stimulates dibasic and neutral amino acid transport and has sequence similarity to glucosidases.

The transport of amino acids across cell membranes is believed to be mediated by integral membrane proteins with distinct substrate specificities. Using expression cloning in Xenopus oocytes and assaying for the uptake of 14C-labeled cystine, we isolated a 2.3-kilobase cDNA (D2) from a rat kidney library. D2 is expressed specifically in kidney and intestine and induces the transport of both neutral and cationic amino acids. The deduced amino acid sequence predicts a 78-kDa protein with a single transmembrane domain, a structure not typical of the known membrane transport proteins, which generally have multiple membrane-spanning regions. The putative extracellular region is highly similar to the 4F2 heavy-chain cell surface antigen and to a family of alpha-glucosidases, which raises the possibility that D2 encodes a transport activator or regulatory subunit.

Amino Acid Sequence↗
PubMed1992

Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin).

The amino acid sequence of galactoglycoprotein purified from human plasma was elucidated to 75% completeness by using chemical degradation of peptides and glycopeptides derived from digests of the protein with seven specific proteases. This sequence represents a polypeptide chain of approximately 220 amino acid residues including a high content of serine, threonine, alanine, and proline with one N-linked and multiple O-linked glycans. Comparison of peptide sequences from the native galactoglycoprotein and the deglycosylated derivative demonstrated the locations of 25 sites of O-glycosylation and three serine sites that are not glycosylated. The homogeneous N terminus was established as serine. C-terminal analysis revealed multiple C-terminal residues, suggesting that galactoglycoprotein molecules are of varying lengths. A search of a protein data base revealed that the galactoglycoprotein polypeptide is identical to the N-terminal (extracellular) polypeptide region of the blood-cell surface molecule CD43 (sialophorin, leukosialin). Further support of the relatedness of these molecules was obtained by immunoprecipitation of 125I-labeled galactoglycoprotein by monoclonal anti-CD43 antibodies. The composition and properties of the molecules together with the known structure of the gene encoding CD43 suggest that galactoprotein is derived by proteolytic cleavage from transmembrane "hexasaccharide CD43," known to be expressed on neutrophils, activated T lymphocytes, and platelets.

Amino Acid Sequence↗
PubMed1992

Cloning of a human kidney cDNA with similarity to the sodium-glucose cotransporter.

We have used low-stringency screening with the human intestinal Na(+)-glucose cotransporter SGLT1 to isolate a 2,271-nucleotide cDNA (Hu14) from human kidney. This clone, which encodes a 672-residue protein, is 59% identical at the amino acid level to SGLT1 and has a similar number and arrangement of predicted membrane-spanning regions. It also shares significant sequence identity with other Na(+)-coupled transporters. Northern blot analysis suggests strong expression of Hu14 in kidney, but, unlike SGLT1, no significant expression in intestine. We have been unable to demonstrate definitive transport of any of a number of substrates (including amino acids, sugars, nucleosides, and vitamins) into Hu14 cRNA-injected Xenopus oocytes, although sequence conservation makes it likely that Hu14 represents another member of the Na+ cotransporter family, possibly a second Na(+)-glucose cotransporter.

Amino Acid Sequence↗
PubMed1992

Expression of mRNA (D2) encoding a protein involved in amino acid transport in S3 proximal tubule.

A rat kidney- and intestine-specific cDNA (D2) that induces high-affinity, Na(+)-independent uptake of cystine and dibasic and neutral amino acids into cRNA-injected Xenopus oocytes was recently isolated by expression cloning in our laboratory (R. G. Wells and M. A. Hediger. Proc. Natl. Acad. Sci. USA 89: 5596-5600, 1992). At present it is not known whether the D2-encoded protein functions as a transporter or as a transporter activator. To gain more insight into the role of D2 in renal amino acid transport, we studied the site of its expression in the kidney. This was determined by Northern blot analysis and by using a combination of in situ hybridization and immunocytochemistry with antibodies that recognize specific proximal tubule segments. D2 antisense RNA hybridized to the same tubular segments that were strongly positive for anti-ecto-adenosinetriphosphatase but negative for carbonic anhydrase type IV and the facilitated glucose transporter GLUT2. We conclude that D2 mRNA is strongly expressed in the rat kidney proximal tubule S3 segment, although there is weak hybridization to the S1 and S2 segments. The signal is absent in all other parts of the kidney. The S3 specific expression of D2 mRNA coincides with the site of high-affinity transport of cystine and other amino acids, consistent with the proposed involvement of D2 in these processes.

Amino Acids↗
PubMed1991

Biosynthesis of the cloned intestinal Na+/glucose cotransporter.

The initial stages in the biosynthesis of the cloned Na+/glucose cotransporter were examined by in vitro expression of the protein in the absence and presence of pancreatic microsomes. Glycosylation was detected by endoglycosidase-H shifts in the apparent size of the proteins on SDS-PAGE. In the presence of microsomes, Mr increased from 52,000 to 58,000, and this was reversed by endo-H. This demonstrates that the protein is glycosylated and that there is no large cleavable signal sequence. Using partial transcripts and site-directed mutagenesis, we established that Asn-248 is glycosylated. Glycosylation was not required for the functional expression of the transporter in Xenopus oocytes. In terms of the topology of the protein, these results suggest that Asn-248 is on the external surface of the membrane.

Acetylglucosaminidase↗
PubMed1991

Intestinal and renal Na+/glucose cotransporters share common structures.

Polyclonal antibodies were raised to peptides selected from three different regions of the cloned rabbit intestinal Na+/glucose cotransporter. Western blot analysis was used to identify the fully mature protein in intestinal and renal brush borders. Two of the antibodies specifically identified a approximately 70-kDa protein band in rabbit intestinal brush borders but did not specifically immunoreact with membranes that do not have Na(+)-dependent glucose transport activity. The immunoreactive proteins had an apparent isoelectric point between pH 4.7 and 6.8. The antibodies also specifically recognized a similar-sized protein in human and seven other mammalian intestinal brush borders. Similar protein bands were identified in four mammalian renal brush-border membranes, indicating shared epitopes between intestinal and renal cotransport proteins. In some species, e.g., lamb and pig, the epitope for one antibody was missing in both intestinal and renal brush borders, suggesting that this epitope is not essential for function. These results suggest that 1) the cloned intestinal Na+/glucose cotransporter is that identified in earlier biochemical studies, 2) there is close structural similarity between intestinal and renal cotransporters, and 3) the structure of these proteins has been conserved during evolution.

Animals↗
PubMed1990

Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells.

Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.

Animals↗