SHORT COMMUNICATION Determination of the Nucleotide Sequence and Chromosomal Localization of the ATP2B2 Gene Encoding Human Ca2+-Pumping ATPase Isoform PMCA2 PAUL BRANDT,1 EDWARD IBRAHIM, GAlL A. P. BRUNS,* AND RACHAEL L. NEVE2 Department of Psychobiology, University of California, Irvine, California 92717; and *Department of Pediatrics, Division of Genetics, The Children's Hospital, Boston, Massachusetts 02115 ReceivedMarch 30, 1992; revised June 26, 1992
The
plasma
membrane
Ca2+-pumping ATPase (Ca2+-ATPase) is responsible f o r m a i n t a i n i n g c a l c i u m h o m e o s t a s i s in eukaryotic c e l l s . T h e C a 2 + - A T P a s e is a family o f p u m p s t h a t a r e e n c o d e d b y at l e a s t four genes. A c D N A for t h e h u m a n v e r s i o n o f C a 2 + - A T P a s e i s o f o r m PMCA2 was i s o l a t e d a n d c h a r a c t e r i z e d . C o m p a r i s o n o f the human and rat cDNA sequences showed that they were 95% homologous in t h e c o d i n g d o m a i n , a n d this h o m o l o g y w a s r e f l e c t e d in t h e d e d u c e d p r o t e i n sequence w h e r e g r e a t e r t h a n 98% h o m o l o g y b e t w e e n t h e human and rat sequences was found. The amino acid d i f f e r e n c e s t h a t w e r e f o u n d w e r e a l m o s t all c o n s e r v a t i v e . T h e P M C A 2 cDNA was used to p r o b e S o u t h e r n b l o t s o f h u m a n - r o d e n t s o m a t i c c e l l hybrid DNAs; t h e results i n d i c a t e d t h a t t h e h u m a n P M C A 2 g e n e w a s loc a t e d o n c h r o m o s o m e 3. ©1992AcademicPress,Inc.
The regulation of free cytosolic calcium levels, essential for proper functioning of all eukaryotic cells, is accomplished by several mechanisms, one of which is the plasma membrane Ca2+-pumping ATPase (Ca2+-ATP ase). The Ca2+-ATPase pumps Ca 2+ from the cytosol to the extracellular space with the concomitant hydrolysis of ATP. The Ca2+-ATPase has been reported to be regulated by a variety of factors, the most well characterized of which are calmodulin, protein kinase C, and cAMPdependent kinase. The action sites of these regulatory factors all occur within a short stretch of amino acids near the carboxyl terminus of the enzyme. Isolation of cDNAs encoding the Ca2+-ATPases has shown that there are at least four distinct genes from The HGM symbol for the plasma membrane Ca2+-transporting ATPase isoform 2 gene is now A T P 2 B 2 . The gene symbol for the plasma membrane Ca2+-transporting ATPase isoform 4 gene, also known as PMCA4, which was mapped to lq25-q32 by Olson et al. (Genomics 9: 629, 1991), is now A T P 2 B 4 . Sequence data from this article have been deposited with the E M B L / G e n B a n k Data Libraries under Accession No. M97260. 1 To whom correspondence should be addressed at Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536-0084. 2 Present address: Molecular Neurogenetics Laboratory, McLean Hospital, Belmont, MA 02178. GENOMICS 14, 484-487 (1992) 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
which different isoforms of the enzyme are derived (2, 3, 6, 7, 9), and each of the primary transcripts can be alternately spliced to yield Ca2+-ATPases with slightly different primary sequences (1, 8). The primarY protein sequence that is changed by alternate RNA splicing is within the regulatory domain of the enzyme. The PMCA1 gene products, PMCAlb, c, and d, contain a cAMP-dependent protein kinase phosphor~;lat~on site within this domain. The P M C A l a isoform, however, lacks this phosphorylation site as a result of a change in the reading frame of the mRNA caused by the insertion of an exon 5' to the sequence encoding the site (8). The PMCA4b isoform does not contain an inhibitory domain near the carboxyl terminus that is found in the PMCA4a isoform. This is due to an exon insertion in the PMCA4b mRNA that alters the reading frame to introduce a stop codon 5' to sequence encoding the inhibitory domain (1). The PMCA1 and PMCA4 cDNAs have been relatively well characterized: cDNA sequences representing the alternately spliced mRNAs have been isolated, and the chromosomal localizations of their respective genes have been determined. In the case of the PMCA2 isoform, however, only one rat cDNA sequence has been determined. To better understand the evolutionary and genetic characteristics of the PMCA2 isoform, we have isolated and sequenced the human cDNA and have localized its genomic location to human chromosome 3. One million recombinant bacteriophage from a human fetal brain cDNA library were screened with an hPMCA4b probe under low stringency, which resulted in the isolation of 28 positive clones. Sequence analysis showed that 27 of these represented spliced forms of PMCA1 and one, HB-17, corresponded to the human equivalent of PMCA2. Because HB-17 did not extend completely in either the 5'- or 3'-direction as judged by comparison with the rat PMCA2 sequence (6), this cDNA clone was used to rescreen the library at high stringency resulting in the isolation of four new cDNA clones. Two of these new isolates, 5'-1 and 3'-1, were characterized further. The positions of the inserts from the three isolates examine~ relative to the complete sequence are diagrammed in Fig. 1. 484
SHORT COMMUNICATION
485
3'-1
E I
5'-1
I E
HB17
E
I
I
5;0
'
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I
.
.
.
.
.
. 2000 .
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hPMCA2 F I G . 1. The hPMCA2 cDNA inserts from ~HB-17, ~5'-1 and X3'-1 are shown with their relative positions to the coding domain. An EcoRI fragment representing bases 2175 to 3580 of hPMCA4b (1) was used to screen a human fetal brain cDNA library at low stringency as described previously (6). The insert from the clone designated XHB-17 was sequenced and found to be the human version of PMCA2. Because this cDNA was not full length, the same human fetal brain library was rescreened at high stringency using the XHB-17 insert as a probe, which yielded clones X5'-I and ~3'-1.
Human Rat
MGDMTNSDFYSKNQRNE S SHGGE FGCTMEELRS LMELRGTEAVVKIKE TYGDTEAI CRRL S S
60
Human Rat
KTS P V E G L P G T A P D L E K R K Q I F G Q N F I P P K K A K P F L Q L V W E A L Q D V T L I I LE IAAI I SLG P T
120
Human Rat
LS F Y H P P G E G N E G C A T A Q G G A E D E G E A E A G W S
180
Human Rat
GLQSRIEQEQKFTVVRAGQVVQI
Human Rat
LTGES DQVRKSVDKDPMLLSGTHVMEGSGRMLVTAVGVNSQTG V
Human Rat
KKAKQQDGA/LAMEMQP LKSAE GGDADDRKKASMHKKEKSVLQGKL K N
Human Rat
AITVI ILVLYFTVDTFVVNKKPWLPECTPVYVQYFVKFFI T
Human Rat
SLAYSVKKMMKDNNLVRHLDACETMGNATAICSDKTGTLTTNRMTVVQAYVGDVHYKEIP L
480
Human Rat
DPS S I N T K T M E L L I N A I A I N S A Y T T K I L P P E K E G A L P R Q V G N K T E C G L L G A L V
540
Human Rat
P V R S Q M P E E K L Y K V Y T F N S V R K S M S TVI K L P D E S F R M Y S K G A S E I V L K K C C K I L N G A G E P 600 M S
Human Rat
R V F R P R D R D E M V K K V I E P M A C E W L R T I C V A Y R D F P S S PE P D W D N E N D I L N E L T C I C V V G I 660 DG
Human Rat
E D P V R P E V P E A I R K C Q R A G I T V R M V T G D N I N T A R A I A I K C G I IH P G E D F L C L E G K E F N R R
720
Human Rat
IRNEKGEIEQERIDKIWPKLRVLARSSPTDKHTLVKGIIDSTHTEQRQVVAVTGDGTNDG
780
Human Rat
PALKKADVGFAMGIAGTDVAKEASDIILTDDNFSSIVKAVMWGRNVYDSISKFLQFQLTV
840
Human Rat
NVVAVIVAFTGACITQDSPLKAVQMLWVNLIMDTFASLALATEPPTETLLLRKPYGRNKP
900
Human Rat
LISRTMMKNILGHAVYQLALIFTLLFVGEKMFQIDSGRNAPLHSPPSEHYTIIFNTFVMM T
960
Human Rat
QLFNEINARKIHGERNVFDGIFRNPIFCTIVLGTFAIQIVIVQFGGKPFSCSPLQLDQWM
1020
Human Rat
WCIFIGLGELVWGQVIATIPTSRLKFLNEAGRLTQKEEIPEEELNEDVEEIDHAERELRR K caM Binding YPKC GQILWFRGLNRIQTQIRVVKAFRSSLYEGLEKPESRTSIHNFFLAHPEFRIEDSQPHIPLI
1080
Human Rat Human Rat
IE G ~ I L L S V I C V V L V T A F N D W S K E K Q F R
PVAE IWGDIAQVKYGDLLPADGLFIQGNDLKIDESS I
240
I IFTLLGAGGEEEEKKD
300
TKLAVQ IGKAGLVMS
360
I G V T V L V V A V P E G L P L A V T I 420
FVLDLKQDYE R
SLETSX DDTDLEEDAALKQNSSPPSSLNKNNSSIDSGINLTTDTSKSATSSSPGSPIHSLETSL A
1140
1190
F I G . 2. Comparison of the deduced protein sequences of human and rat Ca2+-ATPase isoform PMCA2. The human sequence is shown, and only differences between the human and rat sequences are indicated. The regulatory structures and significant conserved sequences are indicated.
486
SHORT COMMUNICATION
C o m p a r i s o n of h u m a n P M C A 2 c D N A sequence to t h e r a t P M C A 2 sequence (6) d i d n o t r e v e a l a n y s i g n i f i c a n t differences b e t w e e n t h e sequences. T h e c o d i n g r e g i o n of t h e c D N A s h o w e d r e m a r k a b l e c o n s e r v a t i o n . As obs e r v e d for h u m a n a n d r a t P M C A 1 (6, 9) a n d h u m a n a n d b o v i n e P M C A 4 (1), t h e h o m o l o g y w i t h i n t h e c o d i n g sequence of h u m a n a n d r a t P M C A 2 was a b o u t 95%. T h i s high degree of h o m o l o g y was n o t f o u n d in t h e 5'-unt r a n s l a t e d regions, however. T h e c o n s e r v a t i o n of t h e m R N A sequence was r e f l e c t e d in t h e d e d u c e d p r o t e i n sequence, w h i c h s h o w e d g r e a t e r t h a n 98% h o m o l o g y bet w e e n h u m a n a n d r a t P M C A 2 s e q u e n c e s (Fig. 2). Of t h e 22 a m i n o acid differences b e t w e e n t h e h u m a n a n d t h e r a t sequences, m o s t were c o n s e r v a t i v e s u b s t i t u t i o n s . Also c o n s e r v e d was t h e sequence S L E T Z , w h e r e X is a h y d r o p h o b i c a m i n o acid, a t t h e c a r b o x y l t e r m i n u s of t h e enzyme. T h i s s e q u e n c e h a s b e e n f o u n d in a l m o s t all isoforms of t h e Ca2+-ATPase, a l t h o u g h its f u n c t i o n h a s n o t been determined. T h e high degree of h o m o l o g y b e t w e e n t h e s a m e isoforms in d i f f e r e n t s p e c i e s - - n o w seen for t h r e e of t h e four k n o w n i s o f o r m s - - s u g g e s t s a r e c e n t e v o l u t i o n a r y event, b u t one t h a t h a d to occur b e f o r e s p e c i a t i o n , since t h e four genes p r o b a b l y e v o l v e d f r o m d u p l i c a t i o n s of an a n c e s t r a l C a 2 + - A T P a s e gene. T h i s s t r o n g c o n s e r v a t i o n of h o m o l o g y w i t h i n t h e s a m e C a 2 + - A T P a s e i s o f o r m families a c r o s s species suggests a s t r o n g selective p r e s s u r e to m a i n t a i n t h e p r i m a r y p r o t e i n sequence of e a c h isoform. T h i s i m p l i e s t h a t e a c h i s o f o r m m u s t h a v e a specific, w e l l - d e f i n e d role in c e l l u l a r f u n c t i o n . T o d e t e r m i n e t h e c h r o m o s o m a l l o c a l i z a t i o n of t h e
ABCDEFGH
TABLE 1 Segregation Pattern of PMCA2 cDNA with DNAs f r o m H u m a n - R o d e n t S o m a t i c Cell H y b r i d s Hybridization signal/ chromosome Chromosome
+/+
-/-
+/-
-/+
Discordant fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x Y
3 2 4 1 2 3 2 2 1 3 2 2 2 3 2 4 1 2 4 4 2 2 0 0
5 4 5 3 3 2 3 2 4 3 3 2 3 2 5 3 3 3 1 1 2 3 4 5
2 1 0 3 2 1 2 2 3 1 2 2 2 1 2 0 3 2 0 0 2 2 4 4
0 0 0 1 2 2 2 3 1 1 2 2 2 2 0 2 2 2 4 4 3 2 1 0
0.22 0.14 0.00 0.50 0.44 0.38 0.44 0.56 0.44 0.25 0.44 0.50 o.44 0.38 0.22 o.22 0.56 0.44 0.44 0.44 0.56 0.44 0.56 0.44
P M C A 2 gene, t h e H B - 1 7 c D N A was h y b r i d i z e d w i t h S o u t h e r n b l o t s of g e n o m i c D N A f r o m h u m a n - r o d e n t cell h y b r i d s as d e s c r i b e d p r e v i o u s l y (4). S e v e n b a n d s were f o u n d to h y b r i d i z e w i t h t h e P M C A 2 p r o b e in t h e h u m a n c o n t r o l D N A s a m p l e (Fig. 3). F o u r of t h e s e b a n d s were c l e a r l y d i s t i n c t f r o m t h e r o d e n t signal a n d a l l o w e d a n u n a m b i g u o u s a s s i g n m e n t of t h e P M C A 2 gene to c h r o m o s o m e 3. T h e s e g r e g a t i o n a n a l y s i s is p r e s e n t e d in T a b l e 1. P r e v i o u s l y , t h e P M C A 1 gene was a s s i g n e d to h u m a n c h r o m o s o m e 12 a n d t h e P M C A 4 gene to chrom o s o m e 1 (5). In n e i t h e r case d i d t h e y m a p to a k n o w n disease m a r k e r . E x a m i n a t i o n of d i s e a s e m a r k e r s on chromosome 3 did not reveal any t h a t would obviously be a s s o c i a t e d w i t h a defect in C a 2 + - A T P a s e f u n c t i o n . T h e a p p l i c a t i o n of h i g h e r r e s o l u t i o n m a p p i n g t e c h niques such as in situ h y b r i d i z a t i o n or R F L P a n a l y s i s to t h e P M C A 2 gene on c h r o m o s o m e 3 m a y e v e n t u a l l y allow a s s o c i a t i o n of t h i s i m p o r t a n t c a l c i u m - r e g u l a t o r y enzyme w i t h an i n h e r i t e d disease. ACKNOWLEDGMENTS This work was funded by NIH Grants NS28406 (R.L.N.) and HD18658 (GAPB).
FIG. 3. Southern blot of human-rodent somatic cell hybrid genomic DNA. Lane A, human genomic DNA control; lane B, cell line A4; lane C, Bh; lane D, D3; lane E, D4; lane F, Dh; lane G, E3; lane I-I,F1; lane I, F3; lane J, G89. Generation of the Southern blot has been described elsewhere (4). The first, third, fifth, and seventh bands in lane A were used in making the assignment of hPMCA2 to chromosome 3.
REFERENCES 1. Brandt, P., Neve, R. L., Kammesheidt, A., Rhoads, R. E., and Vanaman, T. C. (1992). Analysis of the tissue-specific distribution of mRNAs encoding the plasma membrane calcium-pumping ATPases and characterization of an alternately spliced form
S H O R T COMMUNICATION of PMCA4 at the cDNA and genomic level. J. Biol. Chem. 2 6 7 : 4376-4385. 2. Brandt, P., Zurini, M., Neve, R. L., Rhoads, R. E., and Vanaman, T. C. (1988). A C-terminal, calmodulin-like regulatory domain from the plasma membrane Ca2+-pumping ATPase. Proc. Natl. Acad. Sci. USA 85: 2914-2918. 3.
Greeb, J., and Shull, G. E. (1989). Molecular cloning of a third isoform of the calmodulin-sensitive plasma membrane Ca 2+pumping ATPase that is expressed predominantly in brain and skeletal muscle. J. Biol. Chem. 2 6 4 : 18569-18576.
4.
Kosik, K. S., Orecchio, L. D., Bruns, G. A. P., Benowitz, L. I., MacDonald, G. P., Cox, D. R., and Neve, R. L. (1988). Human GAP-43: Its deduced amino acid sequence and chromosomal localization in mouse and human. Neuron 1: 127-132.
5.
Olson, S., Wang, M. G., Carafoli, E., Strehler, E. E., and McBride, O. W. (1991). Localization of two genes encoding plasma membrane Ca2+-transporting ATPases to human chromosomes lq25-32 and 12q21-23. Genomics 9: 629-641.
6.
487
Shull, G. E., and Greeb, J. (1988). Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. J. Biol. Chem. 2 6 3 : 8646-8657. 7. Strehler, E. E., James, P., Fischer, R. Heim, R. Vorherr, T. Filoteo, A. G. Penniston, J. T., and Carafoli, E. (1990). Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane. J. Biol. Chem. 265: 2835-2842. 8. Strehler, E. E., Strehler-Page, M.-A., Vogel, G., and Carafoli, E. (1989). mRNAs from plasma membrane calcium pump isoforms differing in their regulatory domain are generated by alternative splicing that involves two internal donor sites in a single exon. Proc. Natl. Acad. Sc£ USA 86: 6908-6912. 9. Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fisher, R. Heim, R. Vogel, G., Mathews, S., Stehler-Page, M. A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988). Complete primary structure of a human plasma membrane Ca2+-pump. J. Biol. Chem. 2 6 3 : 1415214159.
The
plasma
membrane
Ca2+-pumping ATPase (Ca2+-ATPase) is responsible f o r m a i n t a i n i n g c a l c i u m h o m e o s t a s i s in eukaryotic c e l l s . T h e C a 2 + - A T P a s e is a family o f p u m p s t h a t a r e e n c o d e d b y at l e a s t four genes. A c D N A for t h e h u m a n v e r s i o n o f C a 2 + - A T P a s e i s o f o r m PMCA2 was i s o l a t e d a n d c h a r a c t e r i z e d . C o m p a r i s o n o f the human and rat cDNA sequences showed that they were 95% homologous in t h e c o d i n g d o m a i n , a n d this h o m o l o g y w a s r e f l e c t e d in t h e d e d u c e d p r o t e i n sequence w h e r e g r e a t e r t h a n 98% h o m o l o g y b e t w e e n t h e human and rat sequences was found. The amino acid d i f f e r e n c e s t h a t w e r e f o u n d w e r e a l m o s t all c o n s e r v a t i v e . T h e P M C A 2 cDNA was used to p r o b e S o u t h e r n b l o t s o f h u m a n - r o d e n t s o m a t i c c e l l hybrid DNAs; t h e results i n d i c a t e d t h a t t h e h u m a n P M C A 2 g e n e w a s loc a t e d o n c h r o m o s o m e 3. ©1992AcademicPress,Inc.
The regulation of free cytosolic calcium levels, essential for proper functioning of all eukaryotic cells, is accomplished by several mechanisms, one of which is the plasma membrane Ca2+-pumping ATPase (Ca2+-ATP ase). The Ca2+-ATPase pumps Ca 2+ from the cytosol to the extracellular space with the concomitant hydrolysis of ATP. The Ca2+-ATPase has been reported to be regulated by a variety of factors, the most well characterized of which are calmodulin, protein kinase C, and cAMPdependent kinase. The action sites of these regulatory factors all occur within a short stretch of amino acids near the carboxyl terminus of the enzyme. Isolation of cDNAs encoding the Ca2+-ATPases has shown that there are at least four distinct genes from The HGM symbol for the plasma membrane Ca2+-transporting ATPase isoform 2 gene is now A T P 2 B 2 . The gene symbol for the plasma membrane Ca2+-transporting ATPase isoform 4 gene, also known as PMCA4, which was mapped to lq25-q32 by Olson et al. (Genomics 9: 629, 1991), is now A T P 2 B 4 . Sequence data from this article have been deposited with the E M B L / G e n B a n k Data Libraries under Accession No. M97260. 1 To whom correspondence should be addressed at Department of Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536-0084. 2 Present address: Molecular Neurogenetics Laboratory, McLean Hospital, Belmont, MA 02178. GENOMICS 14, 484-487 (1992) 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
which different isoforms of the enzyme are derived (2, 3, 6, 7, 9), and each of the primary transcripts can be alternately spliced to yield Ca2+-ATPases with slightly different primary sequences (1, 8). The primarY protein sequence that is changed by alternate RNA splicing is within the regulatory domain of the enzyme. The PMCA1 gene products, PMCAlb, c, and d, contain a cAMP-dependent protein kinase phosphor~;lat~on site within this domain. The P M C A l a isoform, however, lacks this phosphorylation site as a result of a change in the reading frame of the mRNA caused by the insertion of an exon 5' to the sequence encoding the site (8). The PMCA4b isoform does not contain an inhibitory domain near the carboxyl terminus that is found in the PMCA4a isoform. This is due to an exon insertion in the PMCA4b mRNA that alters the reading frame to introduce a stop codon 5' to sequence encoding the inhibitory domain (1). The PMCA1 and PMCA4 cDNAs have been relatively well characterized: cDNA sequences representing the alternately spliced mRNAs have been isolated, and the chromosomal localizations of their respective genes have been determined. In the case of the PMCA2 isoform, however, only one rat cDNA sequence has been determined. To better understand the evolutionary and genetic characteristics of the PMCA2 isoform, we have isolated and sequenced the human cDNA and have localized its genomic location to human chromosome 3. One million recombinant bacteriophage from a human fetal brain cDNA library were screened with an hPMCA4b probe under low stringency, which resulted in the isolation of 28 positive clones. Sequence analysis showed that 27 of these represented spliced forms of PMCA1 and one, HB-17, corresponded to the human equivalent of PMCA2. Because HB-17 did not extend completely in either the 5'- or 3'-direction as judged by comparison with the rat PMCA2 sequence (6), this cDNA clone was used to rescreen the library at high stringency resulting in the isolation of four new cDNA clones. Two of these new isolates, 5'-1 and 3'-1, were characterized further. The positions of the inserts from the three isolates examine~ relative to the complete sequence are diagrammed in Fig. 1. 484
SHORT COMMUNICATION
485
3'-1
E I
5'-1
I E
HB17
E
I
I
5;0
'
lo'oo
I
.
.
.
.
.
. 2000 .
ao'oo
I
'
'
'
4o'oo
'
I
4500
hPMCA2 F I G . 1. The hPMCA2 cDNA inserts from ~HB-17, ~5'-1 and X3'-1 are shown with their relative positions to the coding domain. An EcoRI fragment representing bases 2175 to 3580 of hPMCA4b (1) was used to screen a human fetal brain cDNA library at low stringency as described previously (6). The insert from the clone designated XHB-17 was sequenced and found to be the human version of PMCA2. Because this cDNA was not full length, the same human fetal brain library was rescreened at high stringency using the XHB-17 insert as a probe, which yielded clones X5'-I and ~3'-1.
Human Rat
MGDMTNSDFYSKNQRNE S SHGGE FGCTMEELRS LMELRGTEAVVKIKE TYGDTEAI CRRL S S
60
Human Rat
KTS P V E G L P G T A P D L E K R K Q I F G Q N F I P P K K A K P F L Q L V W E A L Q D V T L I I LE IAAI I SLG P T
120
Human Rat
LS F Y H P P G E G N E G C A T A Q G G A E D E G E A E A G W S
180
Human Rat
GLQSRIEQEQKFTVVRAGQVVQI
Human Rat
LTGES DQVRKSVDKDPMLLSGTHVMEGSGRMLVTAVGVNSQTG V
Human Rat
KKAKQQDGA/LAMEMQP LKSAE GGDADDRKKASMHKKEKSVLQGKL K N
Human Rat
AITVI ILVLYFTVDTFVVNKKPWLPECTPVYVQYFVKFFI T
Human Rat
SLAYSVKKMMKDNNLVRHLDACETMGNATAICSDKTGTLTTNRMTVVQAYVGDVHYKEIP L
480
Human Rat
DPS S I N T K T M E L L I N A I A I N S A Y T T K I L P P E K E G A L P R Q V G N K T E C G L L G A L V
540
Human Rat
P V R S Q M P E E K L Y K V Y T F N S V R K S M S TVI K L P D E S F R M Y S K G A S E I V L K K C C K I L N G A G E P 600 M S
Human Rat
R V F R P R D R D E M V K K V I E P M A C E W L R T I C V A Y R D F P S S PE P D W D N E N D I L N E L T C I C V V G I 660 DG
Human Rat
E D P V R P E V P E A I R K C Q R A G I T V R M V T G D N I N T A R A I A I K C G I IH P G E D F L C L E G K E F N R R
720
Human Rat
IRNEKGEIEQERIDKIWPKLRVLARSSPTDKHTLVKGIIDSTHTEQRQVVAVTGDGTNDG
780
Human Rat
PALKKADVGFAMGIAGTDVAKEASDIILTDDNFSSIVKAVMWGRNVYDSISKFLQFQLTV
840
Human Rat
NVVAVIVAFTGACITQDSPLKAVQMLWVNLIMDTFASLALATEPPTETLLLRKPYGRNKP
900
Human Rat
LISRTMMKNILGHAVYQLALIFTLLFVGEKMFQIDSGRNAPLHSPPSEHYTIIFNTFVMM T
960
Human Rat
QLFNEINARKIHGERNVFDGIFRNPIFCTIVLGTFAIQIVIVQFGGKPFSCSPLQLDQWM
1020
Human Rat
WCIFIGLGELVWGQVIATIPTSRLKFLNEAGRLTQKEEIPEEELNEDVEEIDHAERELRR K caM Binding YPKC GQILWFRGLNRIQTQIRVVKAFRSSLYEGLEKPESRTSIHNFFLAHPEFRIEDSQPHIPLI
1080
Human Rat Human Rat
IE G ~ I L L S V I C V V L V T A F N D W S K E K Q F R
PVAE IWGDIAQVKYGDLLPADGLFIQGNDLKIDESS I
240
I IFTLLGAGGEEEEKKD
300
TKLAVQ IGKAGLVMS
360
I G V T V L V V A V P E G L P L A V T I 420
FVLDLKQDYE R
SLETSX DDTDLEEDAALKQNSSPPSSLNKNNSSIDSGINLTTDTSKSATSSSPGSPIHSLETSL A
1140
1190
F I G . 2. Comparison of the deduced protein sequences of human and rat Ca2+-ATPase isoform PMCA2. The human sequence is shown, and only differences between the human and rat sequences are indicated. The regulatory structures and significant conserved sequences are indicated.
486
SHORT COMMUNICATION
C o m p a r i s o n of h u m a n P M C A 2 c D N A sequence to t h e r a t P M C A 2 sequence (6) d i d n o t r e v e a l a n y s i g n i f i c a n t differences b e t w e e n t h e sequences. T h e c o d i n g r e g i o n of t h e c D N A s h o w e d r e m a r k a b l e c o n s e r v a t i o n . As obs e r v e d for h u m a n a n d r a t P M C A 1 (6, 9) a n d h u m a n a n d b o v i n e P M C A 4 (1), t h e h o m o l o g y w i t h i n t h e c o d i n g sequence of h u m a n a n d r a t P M C A 2 was a b o u t 95%. T h i s high degree of h o m o l o g y was n o t f o u n d in t h e 5'-unt r a n s l a t e d regions, however. T h e c o n s e r v a t i o n of t h e m R N A sequence was r e f l e c t e d in t h e d e d u c e d p r o t e i n sequence, w h i c h s h o w e d g r e a t e r t h a n 98% h o m o l o g y bet w e e n h u m a n a n d r a t P M C A 2 s e q u e n c e s (Fig. 2). Of t h e 22 a m i n o acid differences b e t w e e n t h e h u m a n a n d t h e r a t sequences, m o s t were c o n s e r v a t i v e s u b s t i t u t i o n s . Also c o n s e r v e d was t h e sequence S L E T Z , w h e r e X is a h y d r o p h o b i c a m i n o acid, a t t h e c a r b o x y l t e r m i n u s of t h e enzyme. T h i s s e q u e n c e h a s b e e n f o u n d in a l m o s t all isoforms of t h e Ca2+-ATPase, a l t h o u g h its f u n c t i o n h a s n o t been determined. T h e high degree of h o m o l o g y b e t w e e n t h e s a m e isoforms in d i f f e r e n t s p e c i e s - - n o w seen for t h r e e of t h e four k n o w n i s o f o r m s - - s u g g e s t s a r e c e n t e v o l u t i o n a r y event, b u t one t h a t h a d to occur b e f o r e s p e c i a t i o n , since t h e four genes p r o b a b l y e v o l v e d f r o m d u p l i c a t i o n s of an a n c e s t r a l C a 2 + - A T P a s e gene. T h i s s t r o n g c o n s e r v a t i o n of h o m o l o g y w i t h i n t h e s a m e C a 2 + - A T P a s e i s o f o r m families a c r o s s species suggests a s t r o n g selective p r e s s u r e to m a i n t a i n t h e p r i m a r y p r o t e i n sequence of e a c h isoform. T h i s i m p l i e s t h a t e a c h i s o f o r m m u s t h a v e a specific, w e l l - d e f i n e d role in c e l l u l a r f u n c t i o n . T o d e t e r m i n e t h e c h r o m o s o m a l l o c a l i z a t i o n of t h e
ABCDEFGH
TABLE 1 Segregation Pattern of PMCA2 cDNA with DNAs f r o m H u m a n - R o d e n t S o m a t i c Cell H y b r i d s Hybridization signal/ chromosome Chromosome
+/+
-/-
+/-
-/+
Discordant fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x Y
3 2 4 1 2 3 2 2 1 3 2 2 2 3 2 4 1 2 4 4 2 2 0 0
5 4 5 3 3 2 3 2 4 3 3 2 3 2 5 3 3 3 1 1 2 3 4 5
2 1 0 3 2 1 2 2 3 1 2 2 2 1 2 0 3 2 0 0 2 2 4 4
0 0 0 1 2 2 2 3 1 1 2 2 2 2 0 2 2 2 4 4 3 2 1 0
0.22 0.14 0.00 0.50 0.44 0.38 0.44 0.56 0.44 0.25 0.44 0.50 o.44 0.38 0.22 o.22 0.56 0.44 0.44 0.44 0.56 0.44 0.56 0.44
P M C A 2 gene, t h e H B - 1 7 c D N A was h y b r i d i z e d w i t h S o u t h e r n b l o t s of g e n o m i c D N A f r o m h u m a n - r o d e n t cell h y b r i d s as d e s c r i b e d p r e v i o u s l y (4). S e v e n b a n d s were f o u n d to h y b r i d i z e w i t h t h e P M C A 2 p r o b e in t h e h u m a n c o n t r o l D N A s a m p l e (Fig. 3). F o u r of t h e s e b a n d s were c l e a r l y d i s t i n c t f r o m t h e r o d e n t signal a n d a l l o w e d a n u n a m b i g u o u s a s s i g n m e n t of t h e P M C A 2 gene to c h r o m o s o m e 3. T h e s e g r e g a t i o n a n a l y s i s is p r e s e n t e d in T a b l e 1. P r e v i o u s l y , t h e P M C A 1 gene was a s s i g n e d to h u m a n c h r o m o s o m e 12 a n d t h e P M C A 4 gene to chrom o s o m e 1 (5). In n e i t h e r case d i d t h e y m a p to a k n o w n disease m a r k e r . E x a m i n a t i o n of d i s e a s e m a r k e r s on chromosome 3 did not reveal any t h a t would obviously be a s s o c i a t e d w i t h a defect in C a 2 + - A T P a s e f u n c t i o n . T h e a p p l i c a t i o n of h i g h e r r e s o l u t i o n m a p p i n g t e c h niques such as in situ h y b r i d i z a t i o n or R F L P a n a l y s i s to t h e P M C A 2 gene on c h r o m o s o m e 3 m a y e v e n t u a l l y allow a s s o c i a t i o n of t h i s i m p o r t a n t c a l c i u m - r e g u l a t o r y enzyme w i t h an i n h e r i t e d disease. ACKNOWLEDGMENTS This work was funded by NIH Grants NS28406 (R.L.N.) and HD18658 (GAPB).
FIG. 3. Southern blot of human-rodent somatic cell hybrid genomic DNA. Lane A, human genomic DNA control; lane B, cell line A4; lane C, Bh; lane D, D3; lane E, D4; lane F, Dh; lane G, E3; lane I-I,F1; lane I, F3; lane J, G89. Generation of the Southern blot has been described elsewhere (4). The first, third, fifth, and seventh bands in lane A were used in making the assignment of hPMCA2 to chromosome 3.
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S H O R T COMMUNICATION of PMCA4 at the cDNA and genomic level. J. Biol. Chem. 2 6 7 : 4376-4385. 2. Brandt, P., Zurini, M., Neve, R. L., Rhoads, R. E., and Vanaman, T. C. (1988). A C-terminal, calmodulin-like regulatory domain from the plasma membrane Ca2+-pumping ATPase. Proc. Natl. Acad. Sci. USA 85: 2914-2918. 3.
Greeb, J., and Shull, G. E. (1989). Molecular cloning of a third isoform of the calmodulin-sensitive plasma membrane Ca 2+pumping ATPase that is expressed predominantly in brain and skeletal muscle. J. Biol. Chem. 2 6 4 : 18569-18576.
4.
Kosik, K. S., Orecchio, L. D., Bruns, G. A. P., Benowitz, L. I., MacDonald, G. P., Cox, D. R., and Neve, R. L. (1988). Human GAP-43: Its deduced amino acid sequence and chromosomal localization in mouse and human. Neuron 1: 127-132.
5.
Olson, S., Wang, M. G., Carafoli, E., Strehler, E. E., and McBride, O. W. (1991). Localization of two genes encoding plasma membrane Ca2+-transporting ATPases to human chromosomes lq25-32 and 12q21-23. Genomics 9: 629-641.
6.
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Shull, G. E., and Greeb, J. (1988). Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. J. Biol. Chem. 2 6 3 : 8646-8657. 7. Strehler, E. E., James, P., Fischer, R. Heim, R. Vorherr, T. Filoteo, A. G. Penniston, J. T., and Carafoli, E. (1990). Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane. J. Biol. Chem. 265: 2835-2842. 8. Strehler, E. E., Strehler-Page, M.-A., Vogel, G., and Carafoli, E. (1989). mRNAs from plasma membrane calcium pump isoforms differing in their regulatory domain are generated by alternative splicing that involves two internal donor sites in a single exon. Proc. Natl. Acad. Sc£ USA 86: 6908-6912. 9. Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fisher, R. Heim, R. Vogel, G., Mathews, S., Stehler-Page, M. A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988). Complete primary structure of a human plasma membrane Ca2+-pump. J. Biol. Chem. 2 6 3 : 1415214159.
