CDNA CLONING AND IMMUNOLOCALIZATION OF A NA+-H+ EXCHANGER IN LLC-PK1 RENAL EPITHELIAL-CELLS

被引:83
作者
REILLY, RF
HILDEBRANDT, F
BIEMESDERFER, D
SARDET, C
POUYSSEGUR, J
ARONSON, PS
SLAYMAN, CW
IGARASHI, P
机构
[1] YALE UNIV, SCH MED, DEPT INTERNAL MED, NEW HAVEN, CT 06510 USA
[2] YALE UNIV, SCH MED, DEPT CELL BIOL, NEW HAVEN, CT 06510 USA
[3] YALE UNIV, SCH MED, DEPT HUMAN GENET, NEW HAVEN, CT 06510 USA
[4] YALE UNIV, SCH MED, DEPT CELLULAR & MOLEC PHYSIOL, NEW HAVEN, CT 06510 USA
[5] UNIV NICE, CNRS, CTR BIOL, F-06034 NICE, FRANCE
来源
AMERICAN JOURNAL OF PHYSIOLOGY | 1991年 / 261卷 / 06期
关键词
AMILORIDE; SODIUM; INTRACELLULAR PH;
D O I
10.1152/ajprenal.1991.261.6.F1088
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na+-H+ exchangers that can be distinguished by their different sensitivities to the amiloride analogue, N-ethyl-N-isopropylamiloride. In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for one of the exchangers, based on homology with the recently isolated cDNA for a human growth factor-activatable Na+-H+ exchanger (26). There proved to be significant homology between the LLC-PK1 and human sequences, with nucleotide identities of 75, 93, and 85% in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. The LLC-PK1 cDNA encodes a predicted protein of 818 amino acids with a relative molecular mass of 90,999, consisting of an amino-terminal hydrophobic region and a carboxy-terminal hydrophilic region; its deduced amino acid sequence shows 95% identity with that of the human protein. To investigate the localization of the encoded protein, antisera were generated against a synthetic oligopeptide from the hydrophobic region and a fusion protein from the carboxy-terminal hydrophilic domain. Indirect immunofluorescence and confocal microscopy revealed that the antisera labeled the basolateral but not the apical membrane of confluent LLC-PK1 cells. Labeling by the antipeptide antibody was specifically blocked by preincubation with the synthetic peptide and coincided exactly with the pattern produced by a monoclonal antibody against Na+-K+-ATPase. Thus, the LLC-PK1 cDNA encodes the basolateral Na+-H+ exchanger, which must differ structurally from the apical form, at least in the region of the oligopeptide and the fusion protein.
引用
收藏
页码:F1088 / F1094
页数:7
相关论文
共 29 条
[1]   CODON USAGE TABULATED FROM THE GENBANK GENETIC SEQUENCE DATA [J].
AOTA, S ;
GOJOBORI, T ;
ISHIBASHI, F ;
MARUYAMA, T ;
IKEMURA, T .
NUCLEIC ACIDS RESEARCH, 1988, 16 :R315-R402
[2]   SCREENING GAMMAGT RECOMBINANT CLONES BY HYBRIDIZATION TO SINGLE PLAQUES INSITU [J].
BENTON, WD ;
DAVIS, RW .
SCIENCE, 1977, 196 (4286) :180-182
[3]  
BURNS KD, 1990, J AM SOC NEPHROL, V1, P714
[5]   ISOLATION OF BIOLOGICALLY-ACTIVE RIBONUCLEIC-ACID FROM SOURCES ENRICHED IN RIBONUCLEASE [J].
CHIRGWIN, JM ;
PRZYBYLA, AE ;
MACDONALD, RJ ;
RUTTER, WJ .
BIOCHEMISTRY, 1979, 18 (24) :5294-5299
[6]   A TECHNIQUE FOR RADIOLABELING DNA RESTRICTION ENDONUCLEASE FRAGMENTS TO HIGH SPECIFIC ACTIVITY [J].
FEINBERG, AP ;
VOGELSTEIN, B .
ANALYTICAL BIOCHEMISTRY, 1983, 132 (01) :6-13
[7]   IDENTIFICATION OF THE PROTEIN AND CDNA OF THE CARDIAC NA+/H+ EXCHANGER [J].
FLIEGEL, L ;
SARDET, C ;
POUYSSEGUR, J ;
BARR, A .
FEBS LETTERS, 1991, 279 (01) :25-29
[8]   RAPID PRODUCTION OF FULL-LENGTH CDNAS FROM RARE TRANSCRIPTS - AMPLIFICATION USING A SINGLE GENE-SPECIFIC OLIGONUCLEOTIDE PRIMER [J].
FROHMAN, MA ;
DUSH, MK ;
MARTIN, GR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (23) :8998-9002
[9]   PHARMACOLOGICALLY DIFFERENT NA/H ANTIPORTERS ON THE APICAL AND BASOLATERAL SURFACES OF CULTURED PORCINE KIDNEY-CELLS (LLC-PK1) [J].
HAGGERTY, JG ;
AGARWAL, N ;
REILLY, RF ;
ADELBERG, EA ;
SLAYMAN, CW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (18) :6797-6801
[10]   LLC-PK1 MUTANT WITH INCREASED NA+-H+ EXCHANGE AND DECREASED SENSITIVITY TO AMILORIDE [J].
HAGGERTY, JG ;
AGARWAL, N ;
CRAGOE, EJ ;
ADELBERG, EA ;
SLAYMAN, CW .
AMERICAN JOURNAL OF PHYSIOLOGY, 1988, 255 (04) :C495-C501