Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger

Chung Ming Tse, Ann I. Ma, Vincent W. Yang, Alastair J M Watson, Susan Levine, Marshall H. Montrose, James John Potter, Claude Sardet, Jacques Pouyssegur, Mark Donowitz

Research output: Contribution to journalArticle

Abstract

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5′-untranslated region, 2451 bp of open reading frame and 1163 bp of 3′-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90 716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of β-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI - AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.

Original languageEnglish (US)
Pages (from-to)1957-1967
Number of pages11
JournalThe EMBO journal
Volume10
Issue number8
StatePublished - 1991

Fingerprint

Sodium-Hydrogen Antiporter
Cloning
Molecular Cloning
Cell membranes
Complementary DNA
Cell Membrane
Rabbits
Brushes
Microvilli
Amino Acids
Open Reading Frames
Cells
Galactosidases
Kidney Medulla
Glycosylation
Descending Colon
Kidney Cortex
Cell Line
Housekeeping
Proteins

Keywords

  • H exchanger
  • Intestine
  • Na
  • Transfection

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger. / Tse, Chung Ming; Ma, Ann I.; Yang, Vincent W.; Watson, Alastair J M; Levine, Susan; Montrose, Marshall H.; Potter, James John; Sardet, Claude; Pouyssegur, Jacques; Donowitz, Mark.

In: The EMBO journal, Vol. 10, No. 8, 1991, p. 1957-1967.

Research output: Contribution to journalArticle

Tse, CM, Ma, AI, Yang, VW, Watson, AJM, Levine, S, Montrose, MH, Potter, JJ, Sardet, C, Pouyssegur, J & Donowitz, M 1991, 'Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger', The EMBO journal, vol. 10, no. 8, pp. 1957-1967.
Tse, Chung Ming ; Ma, Ann I. ; Yang, Vincent W. ; Watson, Alastair J M ; Levine, Susan ; Montrose, Marshall H. ; Potter, James John ; Sardet, Claude ; Pouyssegur, Jacques ; Donowitz, Mark. / Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger. In: The EMBO journal. 1991 ; Vol. 10, No. 8. pp. 1957-1967.
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abstract = "A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5′-untranslated region, 2451 bp of open reading frame and 1163 bp of 3′-untranslated area, with 70{\%}, 91{\%} and 40{\%} identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90 716) which exhibits 95{\%} amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of β-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI - AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.",
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AU - Ma, Ann I.

AU - Yang, Vincent W.

AU - Watson, Alastair J M

AU - Levine, Susan

AU - Montrose, Marshall H.

AU - Potter, James John

AU - Sardet, Claude

AU - Pouyssegur, Jacques

AU - Donowitz, Mark

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N2 - A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5′-untranslated region, 2451 bp of open reading frame and 1163 bp of 3′-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90 716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of β-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI - AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.

AB - A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5′-untranslated region, 2451 bp of open reading frame and 1163 bp of 3′-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90 716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of β-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI - AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.

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