In an earlier subcellular fractionation study of epithe- lial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphate receptor (IP,R) is ...
THEJOURNAL OF BIOLWICAL CHEMSTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269,No. 38, Issue of September 23,pp. 23694-23699, 1994 Printed in U.S.A.
Epithelial Inositol 1,4,5-Trisphosphate Receptors MULTIPLICITY OF LOCALIZATION, SOLUBILITY, AND ISOFORMS* (Received for publication, May 4, 1994, and in revised form, July 21, 1994) Kevin T. Bush$, Robert 0. Stuart*, Shi-Hua Li§, Luis A. Mouran, AlanH. Sharp§, Christopher A. Ross§, and Sanjay K. NigamSII From the Harvard Medical School, Departments of $Medicine (Renal Division) and Wathology, Brigham and Women's Hospital, Boston, Massachusetts 02115 and §The Johns Hopkins University School of Medicine, Department of Psychiatry and Behavioral Sciences, Baltimore, Maryland 21205
In an earliersubcellular fractionation study of epithelial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphatereceptor (IP,R) is found in association with biochemically distinct cellular membranes, including the endoplasmic reticulum (ER) and plasmamembrane (Sharp, A. H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267,7444-7449).To further characterize epithelial IP,Rs, we have now employed cultured Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Indirect immunofluorescence with an antiserum which specifically recognizes IP,R in MDCK cells by immunoblotting and immunoprecipitationgave an ER-like staining pattern as well as a basolateral plasma membrane-like staining pattern, the latter being particularly evident in highly confluent monolayers. In sections of adult rat kidney tubules a similar staining pattern was observed. Interestingly, whereas known basolateral proteins (Na+,K+-ATPase and the facilitated glucose transporter) gave a continuous basolateral staining pattern, that seenfor IP,Rwas discontinuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggestingthat thepunctate basolateral plasma membrane-likestaining pattern observed for IP,R reflects its localization to basolateral caveolae. Biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP,R and detection with streptavidin, indicated that while most IP,R is localized to biotin-inaccessible compartments (i.e. ER), a fraction (10-20%) of IP,R was accessible to externally added biotin primarily from the basolateral side. Thisresult is compatible withthe dual ER and basolateral caveolar localization suggested by immunocytochemistry, although it does not excludethe presence of some IP,R in the basolateral plasma membrane as well. Solubility studies revealed IP,R to be considerably more insolublethan the basolateral proteins, Na+,K+-ATPase and the liver cell adhesion molecule, as well as the cytoskeletal proteins, ankyrin and fodrin. In the most insoluble fraction, IP,R was found along with caveolin, further supporting the notion that part of the cellular IP,R pool associates with caveolae. Since multiple localizations of IP,R within a cell might reflectthe existence of multiple isoforms, polymerase chain reaction amplificationof first strand cDNA with primers specific for the three isotypes of IP,R was performed. All
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordancewith 18 U.S.C.Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115. Tel.: 617-278-0436;Fax: 617-732-6392.
three isoforms of IP,R were expressedin the homogeneous populationof MDCK cells. The existenceof distinct membrane localizations and multiple isoforms of IP,R within the same cell type suggests an explanation for the complex spatiotemporal patterns of Ca2+release from inositol 1,4,5-trisphosphate-sensitiveCa2+ pools in epithelial and other cells.
The second messenger inositol 1,4,5-trisphosphate UP,),' which is generatedfrom phosphatidyl inositol 4,5-bisphosphate in response to various cellular stimuli(e.g. hormones, growth factors, neurotransmitters), mediates the release of calcium (Ca2+)from intracellular stores upon binding to its receptor (1-3). Although immunocytochemical studies using cerebellar Purkinje cells indicate that theIP, receptor (IP,R) is localized predominantly in rough and smooth endoplasmic reticulum (ER) (4,5-7), experimental evidence strongly suggests that in a variety of non-neural tissues the IP,R exists in association with cellular membranes distinctfrom, although not exclusive of, the ER (8-12). For example, in epithelial tissue (liver and pancreas), we have previously demonstrated by subcellular fractionation thepresence of IP,R in association with both ER and plasma membrane-enriched fractions(11).In addition, recently it was shown that the complex spatiotemporal patterns of Ca2+ release within pancreatic epithelial cells are due, at least in part, toa heterogeneous distribution of IP,-releasable Ca2+pools with differing sensitivities toIP, (13).These findings raise the intriguing possibility that IP,R in epithelial cells might be localized to multiple membrane compartments, each displaying distinct sensitivities to IP,. Furthermore, IP,R is known to exist in multiple isoforms (4, 14-16). The various isoforms appear to be differentially expressed in different tissues (16), although information regarding the expression of IP,Rs in individual cells remains scant. Nevertheless,a multiplicity of IP,R isoforms in epithelialcells could help account for complicated patterns of IP,-mediated Ca2+ release. We have now characterized IP,Rs in Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Our results indicate thatIP,R in individual epithelial cells displays multiple cellularlocalizations, and multipleIP,R isoforms are expressed. These data begin to provide an explanation for the observed spatiotemporal complexity of IP,-mediated Ca2+ release in epithelial cells. The abbreviations used are: IP,, inositol 1,4,54risphosphate; IP,R, inositol 1,4,5-trisphosphate receptor;MDCK, Madin-Darby canine kidney;ER, endoplasmic reticulum; PCR, polymerase chain reaction; LCAM, liver cell adhesion molecule; glut-1, facilitated glucose transporter; PBS, phosphate-bufferedsaline; PAGE, polyacrylamide gelelectrophoresis; bp, base pairb).
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Epithelial IP3 Receptors EXPERIMENTAL.PROCEDURES Reagents a n d Chemicals Antibodies against IP,R purified from rat cerebellum were raised in rabbits andgoats as described previously (11). The initial description of the anti-IP,R sera (APlA and AP2A) used for nearly all studies reported here has been providedelsewhere (11).Antibodies against ribophorin-1 were kindly provided by David Meyer (University of California, Los Angeles), against caveolin by John Glenney (Transduction Laboratories), against Na+,K+-ATPaseby Jonathan Lytton (Haward Medical School),and against the facilitated glucose transporter (glut-1) by Bernard Thorens (University of Lausanne). Secondary antibodies were from Cappel, Boerhinger Mannheim, and the Jackson Laboratories. Zmmunodetection of IP$ Western Blot-Immunoblots of purified rat cerebellar IP,R, MDCK cell lysates, and canine rough and smooth microsomes (prepared as described previously) (11, 17) were probed with antibody against rat cerebellar IP,R and developed using the ECL-chemiluminescent system (Amersham Corp.) with horseradish peroxidase-conjugatedsecondary antisera. The isoform-specificantisera which were tried were raised in rabbits againstthe following peptides based on murine IP,R sequences (16): GHPPHMNVNPQQPA(type I), VSEVSWEILEED (type 111). MDCK Cells-Subconfluent and confluent monolayers of MDCKcells growing on coverslips were fixed by plunging into -80 "C methanol and processed for indirect immunofluorescent localization of several proteins (IP,R (AP2A and APlA), ribophorin-1, Na+,K+-ATPase, and caveolin) as described previously (18, 19). kidneys werefixed in 2% Sections of MouseKidney-Isolated paraformaldehyde in PBS, pH 7.4, for 60 minat room temperature and prepared for cryostat sectioning and indirect immunofluorescentlocalization of IP,R and glut-1 as described previously (20).
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(5'-CTG-GGA-GCT-TTC-AAT-GTC-TGC-3') and IP,R-3'-2 (5'- GCAGTT-CTC-CCC-CGT-CTC-TAC-3'). Primers used to amplify a 182-bp segment between transmembrane domains 2 and 5 of IP,R-I1were M7-5'-3 (5'-GCA-GTG-GCG-ATC-TGC-ACG-TCT-ATG-3') and M7-3'-3 (5'-TCG-GTT-TCC-CAC-AAA-ACT-CAC-CAG-3'). Primers for IP,R-111, which amplified a 160-bp segment between transmembrane domains 6 and7 ofIP,R-111, were M5-5'-3 (5'-CAC-GGA-GCT-GCC-ACA-TTTATG-GGC-3') and M5-3'-3 (5'-TCC-TCA-GTC-CGT-GGT-TCA-TGACGG-3'). Primers for mousep-actin, which amplifieda 350-bp segment, and 3'-1(5'-GTGwere 5'-1(5'-GTT-TGA-GAC-CTT-CAA-CAC-CCC-3')
GCC-ATC-TCC-TGC-TCG-AAG-TC-3'). In each reaction, primers for mouse p-actin were included so that coamplification of actin couldbe used as a control to ensure equal amounts of template and equal efficiencies of amplification in all the samples. Amplification in the absence of template was also used as a control to ensure the products were not the result of DNA contamination. In addition, all samples were amplified using the same master mixture, which contained all components of the reaction. After an initial denaturation stepof 94 "C for 5 min, the cycle was 55 "C annealing for 1min, 72 "C extension for 1min, 94 "C denaturing for 1min, for a total of 26-28 cycles, with a final extension step for 10 min. For each template the PCR reactions were repeated four times. RESULTS
We have previously shown a n association of IP,R with ER and plasma membrane-enriched fractions from epithelial tissue homogenates (11).In addition, a heterogeneous distribution of distinct IP,-releasable Ca2+pools in pancreatic epithelial cells was found in a recent study (13). These data suggest that IP,R in individual epithelial cells has multiple cellular localizations, which might define distinct IP,-sensitive Ca' pools. We therefore characterized IP,Rs in MDCK cells, perhaps the Biotinylation a n d Immunoprecipitation MDCK cells were grown to confluence on 24-mm polycarbonate fil- best studied cultured polarized epithelial cell. When grown in ters (Transwells; Costar) in Dulbecco's minimal essential media (Life culture, thesecells form monolayerswith many characteristics Technologies,Inc.) containing 5% fetal calf serum. After confirmationof of a transporting epithelium and offer an advantageous system tightness (as determined by transepithelial electrical resistance (>200 for studying a homogeneous population of tight polarized epiOhms/cm2)),the monolayers were rinsed twice in PBS containing 1.8 thelial cells (25,26). mM CaCl, and 0.8 mM MgC1, (PBS-CM)and then incubated in PBS-CM To determine whether an affinity-purified serum to rat cercontaining 0.25mg/ml N-hydroxysulfosuccinimide-sulfo-biotinfor20 ebellar IP,R (11)could recognize a similar protein in MDCK min a t 4 "C on either the apical aspect or the basolateral aspect of the cells (21,221. In addition, some filters were identically treated with the cells, we analyzed the specificity of the antibodies on immunoexception that 0.1% Triton X-100 was included topermeabilize the cells. blots andimmunoprecipitates. Purified rat cerebellar IP,R, After rinsing in neutralization buffer (120 mM NaC1, 50 mM "is-HC1, MDCK cell lysates, highly purified canine pancreatic rough pH 7.51, the filters were excised and trimmed of the peripheral 1 mm, microsomes, and smooth microsomes (enriched for plasma and the cells were solubilized in 250 pl of buffer containing 50 m~ Tris-HC1, pH 7.5,250 mM NaCl, 0.2% SDS,5 m~ EDTA, 1mM phenyl- membrane) were subjected to SDS-PAGE, Western blotting, methylsulfonyl fluoride, and 20 pg/ml each of leupeptin, pepstatin A, and probed with affinity-purified antibody against ratcerebeland antipain. Samples were cleared of insoluble material by spinning lar IP,R (APBA,the same antiserum we used previously to for 30 s in a microcentrifuge and brought to a final volume of 1 ml by demonstrate that IP,R fractionated with both ER and plasma addition of buffer containing 50 m~ Tris-HC1, pH 7.5, 2% Triton X-100, membrane) (11).All the samplesshowed a single band at -260 5 mM EDTA, and protease inhibitors. Immunoprecipitates were obkDa when reacted with the antibody (Fig. 1).In addition, imtained by overnight incubation with 4 pl of affinity-purified anti-1P,R serum (AP2A)and 40 pl of protein A-Sepharose CL-4B(1:l w/v slurry of munoprecipitation of permeabilized biotinylated cells resulted affinity matrix). Immunoprecipitates were washed five times in immu- in a single band at -260 kDa, which was shown to be IP,R by noprecipitation buffer, separated by SDS-PAGE, and transferred to ni- immunoblotting (see Fig. 5 ,A and B,discussed below in greater trocellulose. Blots were probed with horseradish peroxidase-conjugated detail). These results indicate that the antibody rabbit raised t o streptavidin andor reprobed with anti-IP,R serum. rat cerebellar IP,R (AP2A)recognizes IP,R in MDCK cells by immunoblotting and immunoprecipitation with apparentspecCell Extraction Confluent monolayers ofMDCK cells were subjected to a modified ificity. Thus, in subsequent experiments, AP2A was the antiTriton X-100 extraction protocol as described previously (18, 23, 24) body primarily used to determine the cellular distribution of which resulted in sequential fractionation of the cells into soluble (S), IP,R in MDCK cells. salt-sensitive cytoskeletal (C) and detergent-insoluble (R) fractions. ImThe cellular distribution of IP,R was examined in both submunoblots of all three fractions (S, C, and R) were prepared, probed confluent (incompletely polarized) and confluent(polarized) with primary antisera (ankyrin, fodrin, LCAM, Na+,K+-ATPase,IP,R, monolayers of MDCK cells. Indirect immunofluorescence of and caveolin) and developed as described above. subconfluentmonolayers of MDCK cells with antibodies Expression of ZP$ in MDCK Cells against rat cerebellar IP,R (AP2A)revealed a perinuclear reTotal RNA was obtained from mouse wholebrain, canine cerebellum, ticular staining pattern extending well into thecytoplasm (Fig. and MDCK cells by the cesium chloride ultracentrifugation method. 2 A ) . This pattern was highly similar (though not identical) to Equal amounts of RNA (10pg) from different tissues were converted to the staining pattern seen with known ER proteins (i.e. ribofirst strand cDNAby RNaseH-reverse transcriptase (Life Technologies, Inc.) with oligo(dt)and random primers. PCR was performedwith 5% of phorin-1; Fig. 2C).This finding, which was obtained with a this reaction product as template with primers specific for each of the different IP,R antibody (APlA) aswell (Fig. 2B),suggests that three types ofIP,R (16). A 447-bp segment between transmembrane IP,R in subconfluent (incompletely polarized) monolayers of domains 3 and of 6 the IP,R-I was amplified using the primers IP,R-5'-2 MDCK cells is associated with theER. The differences in stain-
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ing patternsbetween AP2AIAPlA and ribophorin-1 mayreflect the existence of a distinct ER subcompartment (3, 7 ) or the presence of a small amountof IP,R in a non-ER compartment. In contrast, indirect immunofluorescence of confluent monolayers of MDCK cells with AP2A revealed (in addition to the intracellular ER-like staining pattern)a striking punctate staining near the basolateral plasma membrane (Fig. 3A). The punctate basolateral staining pattern was also obtained with
20516)-1
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t
I I I
I I
RM
M
SM
FIG.1. The anti-IP,R serumAP2A specifically recognizes IP,R on Western blots of MDCK cells. Samples of purified rat cerebellar IP,R, MDCK cell lysates, highly purifiedcanineroughandsmooth microsomes weresubjectedtoSDS-polyacrylamideelectrophoresis, Western blotted and reacted with AP2A a s described under “Experimental Procedures.”AP2Arecognizes a single protein band of -260 kDa in MDCK cells corresponding toIP,R. Lanes from left to right:I, purified rat cerebellar IP,R; M, MDCK cell lysate; R M , highly purified canine rough microsomes; SM, canine smooth microsomes. RM has previously been shown to be highly enriched for rough ER, whereasSM is enriched for plasma membrane, smooth ER, and Golgi (11, 17).
FIG.2. Immunolocalization of IP,R in subconfluent MDCK cells reveals an ER-like staining pattern. SubconfluentmonolayersOTMDCK cells were processed and reacted with two different antisera againstIP,R (A. A P 2 A B. APIA). ribophorin-1 (C),i n d caveolin’(D)a s de: scribed under “Experimental Procedures.” Indirect immunofluorescence demonstrated that these two IP,R antibodies (A and B ) gave a similar staining patterntotheERproteinribophorin-1 ( C ) , while caveolin appeared to have a Golgi-like staining pattern as has been described previously (27,29).
I V’‘
APlA andAPG328,two other IP,R antibodies (data not shown), and was distinct from the more continuous staining pattern seen withknown basolateral proteins(i.e. Na+,K+-ATPase;Fig. 3 0 . This suggested that IP,R might not be localized to true basolateral plasma membrane buta plasma membrane subdomain or an associated membrane compartment. MDCK cells are an in vitro model for polarized epithelia; thus itseemed important to confirm the results inhistological sections of epithelial tissue. Therefore, the intracellular distribution of IP,R was determined inhistological sections of adult rat kidney. Indirect immunofluorescence with AP2A (as well as APlA; data not shown) demonstrated, in addition t o weak intracellular staining, the presence ofIP,R in the basolateral plasma membrane of the polarized epithelial cells comprising the kidney tubule (Fig. 4B).However, the staining pattern obtained for glut-1, a known basolateral membrane protein, although similar, appeared more continuousthan thatobtained for IP,R, and no intracellular staining wasobserved (Fig. 4A). These results indicated that theIP,R localizations we observed in MDCK cells were applicable to polarized epithelial tissue as well. A recentstudy revealedaplasmamembrane-associated IP,R-like protein to be in subplasmalemmel caveolae of endothelium, smooth muscle cells and keratinocytes (12). We therefore sought to determine if the punctate basolateral staining pattern seen with IP,R might reflect its localization to the caveolae of MDCK cells. Indirect immunofluorescence in subconfluent monolayers of MDCK cells with an antibody against the caveolar protein, caveolin, revealed a peri-nuclear Golgilike staining patternas well as some diffuse reticular andpunctate staining (Fig. 20). This result is consistent with other studies which have shown that, while caveolin is present predominantly in cell surface micro-invaginations, it is also localized in endosome-like structures and intubulovesicular structures within the trans-Golgi network (27-29). Thus,the staining patternof caveolin in incompletely polarized (subcon-
Epithelial IP3 Receptors
FIG.3. Immunolocalization of IP,R in confluent MDCK cells reveals both an intracellular (reticular) and a punctate basolateralplasmamembrane s t a i n i n g pattern. Confluent monolayers of MUCK cells were processed and reacted with antiserum againstIP,R (A, APPA), caveolin ( B ) , andNa+,K" ATPase ( C ) as described under "Experimental Procedures." Indirect immunofluorescence revealed IP,R in confluent monolayers of MDCK cells to be present intracellularly, as well as on the hasolateral plasma membrane. The punctate basolateral plasma membrane staining pattern of IP,R (A) is not characteristic of other basolateral plasma membrane proteinslikeNa+,K+-ATPase ( C ) , butis highlysimilartothestainingpattern seenwith the caveolar specific protein, caveolin ( B).
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antigenicity underfixation conditions necessaryto preserve the organellar structures of interest in MDCK cells.) To biochemically confirm the basolateral localization of IP,R and also to obtain an indication of the relative amounts of intracellular IP,R compared with that associated with the cell surface, domain selective biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP,R and detection with labeled streptavidin, was performed (Fig. 5). In the absence of permeabilization, intracellular IP,R should be inaccessible; only cell surface proteinswould be expected to label with biotin (21, 22). Our results indicate that, while most of the IP,R ( S O % of total cellular IP,R) was FIG.4. Immunolocalization of IP,R i n k i d n e y t u b u l e s i s s i m i l alocalized r to biotin-inaccessible sites (Fig. 5A), consistent with to that seen in MDCK cells. Adult rat kidneywas isolated and fixed an ER localization, a significant portion of the IP,R in nonin paraformaldehyde. The kidneys were then frozen, sectioned and reacted with antibodies against glut-1 ( A ) and IP,R ( B ) .Indirect immu- permeabilized cells (200 Ohms/cm2) were biotinylated the apical (first lane), basolateral (second lane), or from both sides in the presence of 0.1% Triton X-100 (th.ird lane). As outlined under “ExperimentalProcedures,” cell lysateswereimmunoprecipitatedwith anti-IP,R serumandproteinA-Sepharose,subjectedto SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidaseconjugated streptavidin. Blots were developed with ECL-chemiluminescent reagents (AmershamCorp.). Quantitation revealed10-20% of the total cellular IP,R (arrowheads)to be accessible to biotinylationat the cell surface (comparison of permeabilized uersus basolateral biotinylation). B , apical and basolateral lanes of blots were reprobed with antiIP,R, revealing that equivalent amounts ofIP,R wereimmunoprecipitated despite the marked difference (basolateral >> apical) inaccessibility to biotin.
idue), which were then subjected to SDS-PAGE and Western blotting (17-18, 24). The cytoskeletal proteins, ankyrin and fodrin, were present in both the soluble and salt-sensitive cytoskeletal fractions, as expected (24) (Fig. 6, lunes S and C). Surprisingly, IP:,R, which was present mainly in the salt-sensitive cytoskeletal fraction and the insoluble residue (Fig. 6, lanes C and R ) ,was much more insoluble than Na+,K+-ATPase or LCAM (Fig. 6), which are known to associate with ankyrin and fodrin (23). Moreover, a substantial fraction of IP3R was found in theinsoluble residue.Under theseconditions, ankyrin and fodrin were nearly completely extracted; thus it difficult is to explain the insolubility of IP,R in thisfraction solely based on the association withankyrin andor fodrin. Moreover, the caveolar-associated protein, caveolin, was also found almost exclusively in this detergent-insoluble residue (Fig. 6, lane R ) . Because of the known insolubility of caveolar proteins (22), it is possible that the fraction of cellular IP,R found in this detergent-insoluble residue represents caveolar IP,R. This interpretation is compatible with the immunocytochemical and biotinylation results. One explanation for the multiple localizations of IP,R within the samecell type (as well as thecomplex solubility properties observed) is the existence of multiple isoforms. At least three IP3Rs have been cloned and sequenced (14-16,32). They appear to have somewhatdifferent tissue distributions and are highly expressed in various regions of the brain and certainepithelia as determinedby Northern blot analysis (16). However, little is known about their expression in individual cell types. To determine whethermultiple IP,Rs were expressedin MDCK cells, cDNA generated from random primed MDCK cell mRNA was subjected to PCR amplification. Since the primers used were based on the sequences of mouse IP,Rs, to investigate the expression of IP,Rs in MDCK (canine) cells, RNAs from dog cerebellum as well as mouse whole brain were used as positive controls. In addition,coamplification of p-actin was used as an internal control to ensure equal amounts of template andequal efficiencies of amplification. Fig. 7 demonstrates that all three
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Frc. 6. Solubility of IP,R in Triton X-100-containing buffers. Confluent monolayers of MDCK cells were subjected to a Triton X-100 extraction protocol which resulted in the sequential fractionation of the cells intosoluble ( S ) , salt-sensitive cytoskeletal ( C ) , and detergentinsoluble ( R ) fractions, which were then dissolved in sample buffer, electrophoresed, and immunoblottedas described under “Experimental Procedures.” From top to bottom, the blots wereprobed with: anti-IP:,R (Ik‘J?); anti-ankyrin (AnK);anti-fodrin (Fod);anti-caveolin (Cau);antiNa+,K+-ATPase( N a K ) ;and anti-LCAM (Lcarn). Note that the basolatera1 cytoskeletal associated proteins Na+,K”ATPase and LCAM were largely in the S fraction, whereas the cytoskeletal proteins ankyrin and fodrin were largely in the C fraction. Caveolin was most insoluble and found predominantly in the R fraction. The solubility properties of IP,R (found in the C and R fractions) were intermediate between ankyrid fodrin and caveolin.
Type I
Type I1
Type 111
“ A B C D A B C D A B C D
FIG.7. PCR analysis of expression of IP,R isoforms in MDCK cells. First strand cDNA was reverse transcrihed from equal amounts of total RNA using oligo(dt) and random primers as described under “Experimental Procedures.” For each experiment, actincDNA and the specific IP,R cDNA were coamplified in the same tube so that actin cDNA could be used as an internal control. In all reactions, the actin band is a t 350 bp. The IP,R isoforms yield bands of: type I, 447 bp; type 11, 182 bp; type 111, 160 bp. All isoforms appear to be expressed in MDCK cells. Lanes: A, mouse brain:R, dog cerebellum; C , MDCK cells; D,no template.
types of IP,R cDNAs could be amplified from the genetically homogeneous population of MDCK cells. In addition, control reactions in each experiment in the absence of template yielded no detectable product. Though this analysis must be viewed as semi-quantitative, the data (relative to actin) suggested that IP,R-I11 was the most abundant subtype in MDCK cells and IP,R-I and IP,R-I1 were expressed in lesser abundance. These data indicate that multiple IP,R isoforms exist in individual epithelial cells and suggest they are not expressed in equal amounts. Furthermore, it consistent is with the possibility that distinct isoforms could be differentially distributed within the same cell type. Nevertheless, of a large number of apparently isoform-specific antisera tested (generated againstpeptide sequences from the mouse cerebellar IP,R isoforms), none appeared to be suitable for immunolocalization in MDCK cells.
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tiple IP,R isoforms with multiple cellular localizations could explain the recently demonstrated patterned IP,-mediated inIn an earlier study, we had identified IP,R in association with tracellular Ca2+changes seen in pancreatic acinar cells (13). subcellular fractions enriched in rough ER and plasma memWhether these multiple IP,Rs are incompartments containing brane obtained from wholetissue homogenates of pancreas and different CaATPases and calcium binding proteins remains to liver (11).In that study, we pointed out that, while the fracbe determined. It is quite possible that, despite the presence of tionation pattern ofIP,R in epithelial tissue was consistent IP,R, the mechanisms of Ca2+regulation in these distinct comwith a dual ER and plasma membrane localization, there were partments could prove to be very different. some differencesin thefractionation patterns of IP3R compared IP3-mediatedsignaling has been implicated in the biogenesis with known ERand plasma membrane proteins, indicating the of tight polarized epithelia (19, 26).Furthermore, MDCK cells need for further immunocytochemical and biochemical charac- are known t o display a complex pattern of increased Ca2+reterization of epithelial IP,Rs. We also discussed the possibility lease during their polarization; the changes in intracellular that multiple IP,R isoforms in epithelial tissues might account Ca2+levels can be highly localized, especiallyat sites of cell-cell for the complex fractionation pattern observed for IP,R. To contact (19). Since cell-adhesion moleculessuch as uvomorulin address some of these issues, we have now employed immuno- are concentrated at basolateral surfaces of epithelial cells, it cytochemical, biochemical,and molecular techniques t o further seems possible that IP,R in basolateral caveolae could play a characterize IP3Rs in a homogeneous population ofMDCK critical role in generating therapid localized intracellular Ca2+ cells, a well studied tight polarized epithelial cell type. changes which appear important for cell adhesive and other In nonpermeabilized MDCK cells, onlya fraction (10-20%) of events critical to polarized epithelial biogenesis (18). the total cellular IP,R was accessible to externally added biotin Finally, although little isknown about the dynamics of caveo(Fig. 51, and thiscell surface IP,R was accessible primarily from lae, the presence of IP,R on a plasma membrane invagination the basolateral side. The result indicates that while most of the that may abut themost peripheral elements of the ER suggests total IP,R in MDCK cells is in a biotin-inaccessible comparta potential mechanism for communication between different ment, as might be expected for a protein limited t o the ER alone, IPpensitive pools within the cell, as well as between the ina significant fraction is incommunication with the cell surface. tracellular pool and the extracellular milieu (1-3, 33). Consistent with the biotinylation data, indirect immunofluoAcknowledgment-We thank Dr. Solomon H. Snyder for critically rescence with a well characterized antibody gave an ER-like staining pattern, as well as a basolateral plasma membrane- reading the manuscript. like staining pattern (Figs. 2-41. However, whereas true basoREFERENCES lateral proteins such as the Na+,K+-ATPaseand the facilitated 1. Berridge, M. J., and Irvine, R. F. (1984) Nature 312,315-321 2. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205 glucose transporter gave a continuous staining pattern at the 3. Lytton, J., and Nigam, S.K. (1992) Cum Opin. Cell Biol. 4, 220-226 cell surface, that seen for IP,R was punctate (Figs. 3 and 4). 4. Mignery, G. A., Newton, C. L., Archer, B. T., 111, and Sudhof, T.C . (1990)J . B i d . This punctate staining was highly similar to that obtained for Chem. 265,12679-12685 5. Maeda, N., Niobe, M., Inoue, Y., andMikoshiba, K., (1989) Deu. Biol. 133, the caveolar protein, caveolin (Fig. 31, suggesting a caveolar 67-76 localization for the cell surface IP,R.Caveolar proteins are 6. Ross, C. A., Meldolesi,J., Milner, T. A., Satoh, T., Supattapone, S., and Snyder, S.H. (1989) Nature 339, 468470 known to be highly insoluble and accessible to cell surface 7. Satoh, T., Ross, C. A., Villa, A,, Supattapone,S., Pozzan, T., Snyder, S.H., and biotin (21, 22, 27-29). Consistent with such a localization, a Meldolesi, J. (1990) J. Cell Biol. 111, 615-624 fraction of IP3Rwas found to be highly insoluble (Fig. 61, in fact 8. Kuno, M., and Gardner, P. (1987) Nature 326,301-304 9. Guillemette, G., Balla, T., Baukal, A. J., and Catt, K. J. (1988) J , Biol. Chem. more insoluble than some basolateral proteins known t o asso263,45414548 ciate with the cytoskeleton. Thus, while association with the 10. Rossier, M. E , Bird, G., and Putney,J. W., Jr. (1991) Biochem. J. 274,643-650 cytoskeleton couldexplain some of the insolubility of IP,R, our 11. Sharp, A. H., Snyder, S.H., and Nigam, S.K. (1992) J. Biol. Chem. 267, 7444-7449 data suggest that another mechanism (e.g. caveolar associa- 12. Fujimoto, T., Nakade, S.,Miyawaki, A,, Mikoshiba, K., and Owaga, K. (1992) tion) could be operative as well. Even so, in principle some of J. Cell Biol. 119, 1507-1514 the IP,R which was labeled by biotin in nonpenneabilized cells 13. Kasai, H., Li, Y. X., and Miyashita, Y. (1993) Cell 74,669-677 14. Danoff, S.IC, Ferris, C. D., Donath, C., Fischer, G. A., Munemitsu, S., Ulrich, could represent IP,R in the trueplasma membrane. NevertheA., Snyder, S.H., and Ross, C . A. (1991)Proc. Natl. Acad. Sci. U. S. A. 88, 2951-2955 less, taken together with our previous fractionation studies T., Okano, H., Furuichi, T., Aruga, J., and Mikoshiba, K. (1991) (111, the datapresented here seem most compatible with a dual 15. Nakagawa, h o c . Natl. Acad. Sci. U. S. A. 88, 62444'248 ER and basolateral caveolar localization. Immunoelectron mi- 16. Ross, C . A., Danoff. S.K., Schell, M. J., Snyder, S. H., and Ulrich, A. (1992) Proc. Natl. Acad. Sci. U. S.A. 8, 4265-4269 croscopy with isoform-specific antibodies will be necessary to 17. Nigam, S. K., and Blobel, G. (1989) J . B i d . Chem. 264, 16927-16932 establish this conclusively. 18. Stuart, R. O., Sun, A., Panichas, M., Hebert, S.C., Brenner, B. M., and Nigam, S. K. (1994) J . Cell. Physiol. 159, 323-333 Multiple localizations forIP,R might be explained by the S. K., Rodriguez-Boulan E., and Silver, R. B. (1992) Proc. Natl. Acad. existence of multiple isoforms within MDCK cells. Although 19. Nigam, Sei. U. S. A. 89, 6162-6166 different IP,R isoforms have been detected in different cell 20. Santos, 0.F. P, Moura, L. A., Rosen, E. M., and Nigam, S. K. (1993)Deu. Bid. 159, 535-548 types, there are little data regarding IP,R isoforms in the same 21. Lisanti, M. P., Tang, Z., and Sargiacomo, M. (1993) J , Cell Biol. 123, 595404 cell type. Thus, PCR amplification ofcDNA generated from 22. Sargiacomo M., Sudol, M., Tang, Z., and Lisanti, M. P. (1993)J. Cell Bid. 122, 789-807 MDCK cells using primers specific for three isoforms of IP,R (IP,R-I, IP,R-I1 and IP,R-111) was performed. The existence of 23. Pasdar, M., and Nelson, W. J. (1988)J. Cell Biol. 106, 677-695 24. Fey, E. G., Wan, K. L., and Penman, S.(1984)J . Cell Biol. 98,1973-1984 all threeisoforms of IP,R was confirmed in these cells (Fig. 7). 25. Rodriguez-Boulan, E., and Nelson, W. J. (1989) Science 245, 718-725 Thus, in this homogeneous epithelial cell line, multiple iso- 26. Nigam, S. K., and Brenner, B. M. (1992) Cum Opin.Nephrol. Hypertension 1, 187-191 forms of IP,R appeared to be present in a single cell. Although 27. Kurzchalia, T. V., Dupree, P., Parton, R. G., Kellner, R., Vina, H., Lehnert, M., our data do not address the question of whether or not each of and Simons, K. (1992) J . Cell Bio2. 118, 1003-1014 the distinct IP,R isoforms identified in epithelial tissue local- 28. Rothberg, K. G., Hewer, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R., and Anderson, R. G. W. (1992) Cell 68, 673-682 izes to distinct membrane compartments (a number of different 29. Dupree, P., Parton, R. G., Raposo, G., Kurzchalia, T. V., and Simons, K. (1993) EMBO J . 12.1597-1605 isofom-specific antisera raised againstmurine peptide seNelson, W. J., LdHammerton, R. W. (1989) J . Cell Biol. 108, 893-902 quences that we tried were not reactive in MDCK cells, possibly 30. 31. Nelson, W. J., Shore, E. M., Wang, A. Z., and Hammerton, R. W. (1990)J . Cell B i d . 110, 349-357 due to interspecific differences and/orlow levels of expression), T. C . , Newton, C . L., Archer 111, B. T.,Ushkaryov, Y. A., and Mignery, these data suggest a differential subcellular distribution of 32. Sudhof, G.A.(1991) EMBO J . 10,3199-3206 IP3R isoforms within individual epithelial cells. Moreover,mul- 33. Putney, J. W., and Bird, G. St. J. (1993) Cell 75,199-201 DISCUSSION
