1 title: evidence that type i, ii, and iii inositol 1,4,5-trisphosphate

1

Title: Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as

integral plasma membrane proteins.

Akihiko Tanimura*, Yosuke Tojyo* and R. James Turner**.

*Department of Dental Pharmacology, School of Dentistry, Health Sciences University of

Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.**Gene Therapy and Therapeutics

Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.

Correspondence to:

Name: Dr. Akihiko Tanimura

Tel: +81-1332-3-1211

Fax: +81-1332-3-1399

E-mail: [email protected]

Running title: IP3 receptors on the plasma membrane

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 28, 2000 as Manuscript M004495200 by guest on January 29, 2018

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Summary

A number of previous reports have suggested that inositol 1,4,5 trisphosphate

receptors (IP3R's) are present in the plasma membranes of cells. We confirm this directly in

the present study by demonstrating that a significant proportion of the IP3R's found in A431

cells, Jurkat cells and rat parotid acini can be biotinylated by the extracellular application of

sulfo-NHS-biotin to intact cells. This labeling cannot be accounted for by the reaction of

sulfo-NHS-biotin with intracellular IP3R's since calnexin and the SERCA2 ATPase, both

integral membrane proteins of the endoplasmic reticulum, are not labelled under the same

experimental conditions. Individual IP3R subtypes were detected using subtype specific

antibodies. A431 cells expressed only the type-3 IP3R and 23% of this protein was in the

biotinylated (plasma membrane) fraction. Jurkat cells and rat parotid cells expressed all three

IP3R subtypes. Contrary to earlier results suggesting that only the type-3 IP3R might localize

to the plasma membrane, we found that significant amounts (5-14%) of all three subtypes

could be identified in the biotinylated fractions of Jurkat and rat parotid cells. Our results

suggest a role for IP3R's in plasma membrane as well as intracellular membrane function.

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Introduction

A wide variety of hormones, neurotransmitters, growth factors, and other

physiological and pathological stimuli result in increased phosphoinositide turnover and

mobilization of Ca2+ from intracellular stores. In this process, IP3 plays a central role by

activating IP3 receptors (IP3R’s) on the endoplasmic reticulum. These receptors are IP3-gated

Ca2+ channels (1) that generate Ca2+ signals by releasing Ca2+ from luminal stores (2). In

mammalian cells, there are at least three IP3R subtypes, type-1 (IP3R-1), type-2 (IP3R-2) and

type-3 (IP3R-3), though the actual role of each subtype in physiological Ca2+ signaling

remains to be established. IP3-induced Ca2+ depletion of intracellular stores is typically

followed by the activation of a Ca2+ influx pathway across the plasma membrane, a process

known as “capacitative Ca2+ entry”. Considerable recent research has focussed on

homologues of the Drosophila mutant, trp, as candidates for this Ca2+ entry channel and on

the mechanism whereby IP3R's and/or the depletion of intracellular Ca2+ stores trigger the

capacitative Ca2+ entry step. However, in addition to the well-established role of IP3R's as

Ca2+ release channels on the endoplasmic reticulum, there is also evidence suggesting the

presence of functional IP3 receptors on the plasma membrane (3, 4).

IP3-dependent Ca2+ entry across the plasma membrane was first characterized

electrophysiologically by Kuno and Gardner (5). They reported that the application of IP3 to

excised patches of the plasma membrane of Jurkat cells activated an inward current. Similar

results have been reported in variety of other cells including the A431 human carcinoma cell

line (6, 7), vascular endothelial cells (8), and olfactory receptor neurons (9, 10).

Immunolabelling studies in several cell types have also provided evidence that a portion of

the cellular IP3R pool is localized to the plasma membrane (11-13). In related experiments,

DeLisle et al (14) observed that IP3R-3, but not IP3R-1, was detected at or near the plasma

membrane when these proteins were expressed in Xenopus laevis oocytes. These authors also

found that expression of IP3R-3, but not IP3R-1, markedly increased the magnitude and

duration of IP3-induced Ca2+ influx. A number of subcellular fractionation studies have also

found that IP3R's often appear in the plasma membrane fraction (15-18).

The results of several recent studies indicate that a close association between the


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endoplasmic reticulum and the plasma membrane is required to activate capacitative Ca2+

entry (19-22). This association may be required for the insertion of channels or regulatory

molecules into the plasma membrane (21, 22) or for the direct interaction of IP3R's with the

capacitative Ca2+ entry channel (19, 20) as first proposed in the “conformational coupling”

model of Irvine (3, 23). These studies raise the possibility that some of the observations

suggesting the presence of IP3R's in the plasma membrane could be explained by the

association of the endoplasmic reticulum (and thereby IP3R's) with the membrane, rather than

by the actual presence of IP3R's as integral plasma membrane proteins. While it seems

unlikely that all of these diverse observations can be explained in this way, it is obviously

important to resolve this question of IP3R localization in order to fully understand the role of

these proteins in Ca2+ signalling and possibly other processes. In the present paper we employ

the membrane-impermeant biotinylating reagent, sulfo-NHS-biotin (24) to directly label and

isolate plasma membrane proteins from several cell types. Using antibodies specific for each

of the three IP3R subtypes we provide strong evidence that each of these subtypes can occur

as integral plasma membrane proteins.


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Experimental Procedures

Materials

Sulfo-NHS-biotin, immobilized protein G-agarose, peroxidase-conjugated goat anti-

rabbit antibody and peroxidase-conjugated avidin (avidin-HRP) were purchased from Pierce

(Rockford, IL, USA). Tosyl-L-phenylalanine chloromethyl ketone (TPCK) was obtained from

Boehringer Mannheim. Immobilized avidin-agarose beads (avidin-beads), thyroglobulin, and

soybean trypsin inhibitor were from Sigma. Phenylmethylsulfonyl fluoride (PMSF), pepstatin

A, leupeptin, and dithiothreitol (DTT) were from Wako Pure Chemicals (Osaka, Japan).

Peroxidase-conjugated goat anti-mouse antibodies were obtained from Biosource

International (Camarillo, CA, USA). Tris-glycine SDS sample buffer was from Novel

Experimental Technology (San Diego, CA, USA). Anti-calnexin monoclonal antibody was

purchased from Transduction Laboratory (Lexington, KY, USA). Anti-SERCA2 ATPase

monoclonal antibody was from Affinity Bioreagents (Golden, CO, USA)

Protein concentration was measured with the BCA protein assay system (Pierce).

Anti IP3 Receptor Antibodies

Polyclonal antibodies were raised against C-terminal peptides specific to each of the

three IP3R subtypes using a strategy very similar to that of Wojcikiewicz (25). The anti-IP3R-

1 antibody was raised against the peptide RIGLLGHPPHMNVNPQQPA corresponding to

amino acids 2733-2751 of the human IP3R-1. Exactly the same sequence is present in rat

IP3R-1. The anti-IP3R-2 and anti-IP3R-3 antibodies were raised against the peptides

CGFLGSNTPHVNHHMPPH and RLGFVDVQNCISR corresponding to amino acids 2685-

3001 and 2659-2671 of the human IP3R-2 and IP3R-3, respectively (an N-terminal cysteine

was added to the former peptide to facilitate coupling reactions). The corresponding

sequences in rat IP3R-2 and IP3R-3 differ by a single amino acid in each case. Polyclonal

antisera were raised against these peptides coupled to keyhole limpet hemocyanin by Genosys

Biotechnologies, Inc. (Woodlands, TX, USA). Crude antisera were then affinity-purified

against their corresponding peptides coupled to EDC/Diaminodipropylamine (IP3R-1 and

IP3R-2 peptides) or SulfoLink (IP3R-3 peptide) columns (Pierce). The affinity-purified


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antibodies against IP3R-1, IP3R-2 and IP3R-3 are referred to as ABsI, ABsII, and ABsIII,

respectively, in this paper.

In preliminary experiments, the dilutions of ABsI, ABsII, and ABsIII were adjusted

so that IP3R-1, IP3R-2, and IP3R-3 produced bands of equal intensity in Western blots of rat

lung since this tissue has been reported to expresses all three IP3R subtypes at approximately

the same levels (25).

Cell Culture

The A431 human carcinoma cell line was obtained from Riken gene bank (Tokyo,

Japan). The Jurkat human T cell line was a generous gift from Dr. K.Tamoto (Health Sciences

University of Hokkaido, Hokkaido, Japan). A431 cells were plated in 100-mm-diameter

dishes at a concentration of 1x106 cells per dish in Dulbecco's modified Eagle's medium

(Gibco/BRL) with high glucose (25mM), supplemented with 10% fetal bovine serum, 4mM

L-glutamine, 1mM sodium pyruvate, penicillin (100 unit/ml), and streptomycin (100 µg/ml).

A431 cells were used for experiments six days after plating (80-90% confluent). Jurkat cells

were plated at a concentration of 1x 105 cell per dish in RPMI 1640 medium (Gibco/BRL),

supplemented with 10 % heat-inactivated fetal bovine serum, penicillin (100 unit/ml), and

streptomycin (100 µg/ml). Jurkat cells were used for experiments three days after the plating.

Parotid acinar preparation

Male Wister strain rats (2-3 mo) were anesthetized with diethyl ether and killed by

cardiac puncture. Parotid grands were minced and digested with trypsin and collagenase as

described elsewhere (26). The dispersed rat parotid cells were washed and resuspended in

Hanks' balanced salt solution buffered with 20 mM Hepes-NaOH, pH 7.4 (HBSS-H).

Biotinylation of cell surface proteins

Jurkat cells and rat parotid acinar cells were washed with PBS/Ca/Mg (PBS

containing 2mM CaCl2 and 1mM MgCl2) and collected by centrifugation at 400 x g for 3 min.

These cells were biotinylated with a 30 min-incubation in PBS/Ca/Mg containing 0.5 mg/ml


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sulfo-NHS-biotin at 4 oC. A431 cells adherent to the culture dish were washed with

PBS/Ca/Mg and incubated for 30 min in PBS containing 0.5 mg/ml sulfo-NHS-biotin at 4 oC.

In all cases the reaction of sulfo-NHS-biotin was terminated by adding 100mM glycine and

washing with PBS/Ca/Mg. The biotinylated cells were then resuspended in homogenization

buffer A (HB-A; PBS containing 200µM PMSF, 10 µM pepstatin A, 100 µM TPCK, 1mM

DTT, 10 µM leupeptin, 0.1 mg/ml soybean trypsin inhibitor) and disrupted for 2 min in a

sonicator bath (Model 2510J-MT, Branson Ultrasonic Corp., Danbury, CT, , USA). Disrupted

cells were centrifuged at 40,000 x g for 20 min, and the resulting particulate fraction was

resuspended in HB-A and stored at -80 oC until use. These preparations are referred to as

“externally-biotinylated” cell samples in the paper.

In some experiments, cells were disrupted with a Polytron homogenizer (40 sec x 2)

in 2 ml of PBS/Ca/Mg, mixed with 2 ml of PBS containing 1mg/ml sulfo-NHS-biotin (final

concentration 0.5mg/ml), and incubated for 30 min at 4 oC. After terminating the reaction by

adding 100 mM glycine, the homogenate was centrifuged at 40,000g for 20 min. The

particulate fraction was resuspended in HB-A and recentrifuged at 40,000g for 20 min. The

resulting particulate fraction was resuspended in HB-A and stored at -80 oC until use. These

preparations are referred to as “biotinylated-homogenates” in the paper.

Isolation of biotinylated proteins

Externally-biotinylated cell samples (450µg protein/150µl) were solubilized in HB-A

containing 1% Triton X-100 (HB-A-TX) for 10 min and centrifuged at 40,000 x g for 15 min.

The supernatant was mixed with 30 µl of avidin-beads and immediately centrifuged at 400 x g

for 20 sec. A 50 µl aliquot of the supernatant was taken as the “original extract”. The rest of

mixture (100 µl sample plus 30 µl avidin-beads) was incubated for 2 hr at 4 oC, then

centrifuged at 400 x g for 20 sec. The supernatant (100 µl) was taken as the “avidin-

unbound“ fraction. The collected beads were washed twice with HB-A-TX, twice with 1.5 M

Guanidine-HCl, and twice again with HB-A-TX. Protein retained by the washed beads was

then eluted with 100 µl of electrophoresis sample buffer (“avidin-bound” fraction). A similar

procedure was used for biotinylated-homogenates except that the protein concentration of the


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sample was decreased to 30 µg /150 µl and the volume of avidin-beads was increased to 100

µl. In these experimental protocols, ~90% of biotinylated proteins were recovered in the

“avidin-bound” fraction (not shown).

Microsomal preparation from cells and rat tissues

A431 cells on a culture dish were washed with HBSS-H and scraped into ice-cold

homogenization buffer B (HB-B) containing 93.5mM trisodium citrate, 6.2mM citrate,

200µM PMSF, 10 µM pepstatin A, 100 µM TPCK, 1mM DTT, 10 µM leupeptin, and 0.1

mg/ml soybean trypsin inhibitor. The cells were then disrupted with a Polytron homogenizer

(10 sec x 2). Jurkat cells and rat parotid cells were washed with HBSS-H and collected by

centrifugation at 400 x g for 3 min. Cell pellets were then resuspended in ice-cold HB-B and

disrupted with the Polytron homogenizer (20 sec x 2). Rat tissues (cerebellum, lung and

liver) were dissected from male Wister strain rats. Minced tissues were rinsed with HBSS-H

and disrupted in HB-B (30 sec x 2 with a Polytron homogenizer). Microsomal fractions were

then prepared as follows: The above homogenates were centrifuged at 1,500 x g for 10 min

and the resulting pellet was discarded. The supernatant was centrifuged at 40,000 x g for 20

min and the resulting microsomal pellet was resuspended in HB-B and kept at -80 oC until

use.

Immunoprecipitaion of IP3R’s

Externally-biotinylated cell samples (1mg protein/ml) or microsomal fractions (1-2mg

protein/ml) were solubilized in HB-B with 1% Triton X-100 (HB-B-TX) for 10 min and

centrifuged at 40,000 x g for 15 min at 4 oC. A 200-400 µl aliquot of the supernatant was

incubated for 2 hr at 4 oC with either ABsI, ABsII, or ABsIII (10 µl) in the presence of 2%

ovalbumin. Protein G-agarose beads (10 µl), prewashed with HB-B-TX containing 2%

ovalbumin, were then added to the sample-antibody mixture. After 1 hr of additional

incubation, the beads were collected by centrifugation and washed five times with HB-B-TX

changing the tube for the last spin. Immunoprecipitated protein retained by the washed beads

was then eluted with 50 µl of electrophoresis sample buffer.


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Western blot analysis

Samples were subjected to electrophoresis on 3-8% NuPAGE Tris-Acetate gels

(Novel Experimental Technology), then transferred to nitrocellulose (Schleicher & Schuell,

Dassel, Germany) or Immobilon-P (Millipore) membranes. Nitrocellulose membranes were

blocked for one hour with Block Ace (Dainippon Pharmaceuticals, Osaka, Japan) then

incubated for 2 hours with affinity purified ABsI (300 ng/ml), ABsII (120 ng/ml) or ABsIII

(100 ng/ml), as appropriate, in 10% Block Ace. Blots were then incubated for 1 hour with

peroxidase-conjugated goat anti-rabbit IgG (80 ng/ml) in 10% Block Ace. For detection of

calnexin, Immobilon-P membranes were blocked for one hour in Tris buffered saline

containing 4% skim milk powder (TBS/SM) then incubated for 2 hours with anti-calnexin

monoclonal antibody (250 ng/ml) in TBS/SM. These blots were then incubated for 1 hour

with peroxidase-conjugated goat anti-mouse IgG (100 ng/ml) in TBS/SM. For detection of

SERCA2 ATPase, Immobilon-P membranes were blocked for one hour in PBS containing 3%

BSA (PBS/BSA) then incubated for 2 hours with anti-SERCA2 ATPase monoclonal antibody

(1:2500 dilution) in PBS/BSA. These blots were then incubated for 1 hour with peroxidase-

conjugated goat anti-mouse IgG (100 ng/ml) in PBS/BSA. Visualization of immunoreactive

bands was carried out using the ECL kit (Amersham Pharmacia Biotech).

To detect biotinylated proteins, blots were incubated for 1 hr with avidin-HRP

(2µg/ml) in TBS/SM. Reactive bands were detected using the ECL kit.

Prestained SDS-PAGE molecular weight markers (BioRad Laboratories) were run on

each blot. The molecular weights of these markers as well the molecular weights of the IP3R’s

and calnexin were determined using the HMW-SDS Marker Kit from Amersham Pharmacia

Biotech (Mr=53,000-212,000) and thyroglobulin (Mr=330,000).

Data Analysis

Quantitative analysis of Western blots was carried out using the program NIH Image

after digital scanning of the exposed X-ray films. All quantitative data are quoted as mean ±


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S.E. (number of experiments). Results shown in all Figures are representative of at least three

independent analyses.


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Results

Characterization of Antibodies

Fig. 1A shows the results of Western blots carried out on rat lung microsomes using

our three antipeptide antibodies raised against the C-termini of IP3R-1, IP3R-2, and IP3R-3

(ABsI, ABsII, and ABsIII, respectively; see Methods). As already mentioned, rat lung has

been shown to express all three IP3R subtypes (25). ABsI labels a single band at ~270 kDa,

while ABsII and ABsIII both label bands at ~260 kDa; these apparent molecular weights are

in good agreement with those previously reported for IP3R-1, IP3R-2 and IP3R-3 (25, 27). To

confirm that each of these antibodies recognizes a distinct protein, we carried out

immunoprecipitations with ABsI from rat cerebellum, with ABsII from rat liver, and with

ABsIII from A431 cells (rich sources of IP3R-1, IP3R-2 and IP3R-3, respectively (25, present

paper). The immunoprecipitated material was then analysed by Western blotting with each of

the three antipeptide antibodies (Fig. 1B). It is clear from Fig. 1B that none of these

antibodies cross reacts with the protein immunoprecipitated by either of the two others. Taken

together with the fact that each of these antibodies was raised and purified against a peptide

specific to one of the IP3R subtypes, these results provide strong confirmation of their IP3R

binding and subtype specificity.

Evidence that IP3R-3 is present in the plasma membrane of A431 cells

As illustrated in Fig. 2A, IP3R-3 is the only IP3R subtype detectable in A431 cells. In

order to determine whether IP3R-3 was present in the plasma membrane of these cells we

selectively biotinylated their external surface with the membrane-impermeant amino reagent,

sulfo-NHS-biotin (see Methods). In control experiments (not shown) using the criterion of

trypan blue exclusion we found that >99% of these sulfo-NHS-biotin-treated cells were

viable. Fig. 2B shows that ABsIII immunoprecipitated IP3R-3 from these externally-

biotinylated cells as expected (lane 1). This immunoprecipitation was prevented in the

presence of the IP3R-3 C-terminal peptide against which ABsIII was raised (lane 2), further

confirming the specificity of the antibody interaction. Immunoblotting of the same samples

with avidin-HRP revealed a biotinylated protein with the same apparent molecular weight as


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IP3R-3 (lane 3). This protein was likewise not observed when immunoprecipitation was

carried out in the presence of the IP3R-3 C-terminal peptide, consistent with the conclusion

that this biotinylated protein is indeed IP3R-3. Since only plasma membrane proteins exposed

to the extracellular solution are expected to be labelled by sulfo-NHS-biotin under our

experimental conditions, these results strongly suggest that IP3R-3 is present in the plasma

membrane of A431 cells.

To further confirm the presence of IP3R-3 in the plasma membrane, we carried out a

reverse experiment to the one just described; more specifically, we precipitated biotinylated

proteins from externally-biotinylated A431 cells using avidin beads, then assayed this

material for IP3R-3 using ABsIII. The results of such an experiment are illustrated in Fig. 2C.

We first demonstrate that the level of ABsIII immunoreactivity is similar in extracts from

sulfo-NHS-biotin-treated (lane 1) and sulfo-NHS-biotin-untreated (lane 2) cells before avidin

precipitation. Next we show that a portion of this starting immunoreactivity is observed in the

avidin-bound fraction from biotinylated cells (lane 3). But this signal is not seen when avidin-

specific precipitation is blocked by the presence of excess added biotin (lane 4) or when

avidin precipitation is carried out on non-biotinylated cells (lane 5). These results strongly

support the conclusion from Fig. 2B that IP3R-3 is biotinylated by the extracellular

application of sulfo-NHS-biotin to A431 cells.

Evidence that sulfo-NHS-biotin does not label intracellular sites and quantitation of plasma

membrane associated IP3R-3 in A431 cells

In order to verify that extracellular sulfo-NHS-biotin does not label intracellular sites

in intact A431 cells we assayed for the possible biotinylation of calnexin, a 90 kDa integral

membrane protein known to be localized to the endoplasmic reticulum (28). In these

experiments biotinylated proteins from externally-biotinylated A431 cells were isolated on

avidin beads and samples of the original extract, the avidin-bound fraction and the avidin-

unbound fraction, were analysed by immunoblotting with anti-calnexin antibody. As

illustrated in Fig. 3A no anti-calnexin immunoreactivity was detectable in the avidin-bound

fraction, while the same material probed with ABsIII produced a readily observable signal


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(Fig. 3B). To confirm that calnexin could be labelled by sulfo-NHS-biotin in permeabilized

cells, we repeated these assays on biotinylated homogenates of A431 cells (see Methods). In

complete contrast to the result with intact cells, in this case all of the anti-calnexin

immunoreactivity was associated with the avidin-bound fraction (Fig. 3C). A similar result

was observed with ABsIII (Fig. 3D) as expected. Essentially identical results to those

obtained in Figs. 3A and 3C were found when these same fractions were probed with

antibody against the SERCA2 ATPase (see Methods; results not shown), which like calnexin

is also an integral membrane protein of the endoplasmic reticulum. These experiments

provide strong evidence that intracellular proteins such as calnexin and the SERCA2 ATPase

are not biotinylated by the application of extracellular sulfo-NHS-biotin to intact A431 cells,

and thus that the biotinylation of IP3R-3 demonstrated in Figs. 2B, 2C and 3B is due to

labelling by sulfo-NHS-biotin on an extracellular site. Since IP3R-3 is an integral membrane

protein these results strongly suggest that IP3R-3 is an integral membrane protein of the A431

cell plasma membrane.

From the results shown in Fig. 3B we can determine the proportion of IP3R-3 found

in the avidin-bound fraction by quantitative analysis of the various immunoreactive bands

(see Methods). In this particular experiment we find that the intensities of the IP3R-3 band in

lanes 2 and 3 are 75% and 20%, respectively, of that in lane 1. To confirm the reliability of

this quantitative result we have also run a three-fold larger sample of the avidin-bound

material in lane 4. The intensity of this band is 50% of that in lane 1, indicating this procedure

is reasonably accurate. The combined analyses of 5 experiments like the one shown in Fig. 3B

indicate that approximately 23% of the IP3R-3 expressed in A431 cells can be biotinylated by

extracellular sulfo-NHS-biotin and therefore is found in the plasma membrane of these cells

(Table 1).

IP3R subtypes in the plasma membranes of Jurkat cells and rat parotid cells.

The experiments described above for A431 cells were next extended to Jurkat cells

(Fig. 4) and dispersed rat parotid acini (Fig. 5). In Figs. 4A and 5A we demonstrate that each

of these cell-types expresses all three IP3R subtypes, albeit in somewhat differing proportions.


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When these cells were externally-biotinylated and the resulting material was analysed for

biotinylated IP3R's as in Fig. 3B we found that significant amounts of all three IP3R subtypes

were present in the avidin-bound fraction for both cell-types (Figs. 4B and 5B, and Table 1).

When the same material was probed for calnexin immunoreactivity we found, as for A431

cells (Fig. 3B), that all of this activity was associated with the avidin-unbound

(unbiotinylated) fraction. Similar results to those for calnexin were found when samples from

Jurkat cells were probed with anti-SERCA2 ATPase antibody (results not shown; this

antibody does not cross-react with rat SERCA2 ATPase and thus could not be used to probe

rat parotid samples). Control experiments using the criterion of trypan blue exclusion

indicated that the viabilities of sulfo-NHS-biotin-treated Jurkat and rat parotid acini were 98.5

± 0.2 % (n=6) and 93.8 ± 0.9 % (n=5), respectively.


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Discussion

The present study provides strong evidence that a significant proportion of the IP3R's

found in A431 cells, Jurkat cells and rat parotid acini can be biotinylated by the extracellular

application of sulfo-NHS-biotin to intact cells. This labelling cannot be accounted for by the

reaction of sulfo-NHS-biotin with intracellular IP3R's since calnexin and the SERCA2

ATPase, both integral membrane proteins of the endoplasmic reticulum, are not labelled

under the same experimental conditions. While it is conceivable that our results could be

explained by the coprecipitation of IP3R’s with an associated biotinylated protein, we feel

that this possibility is quite remote. Such a protein would not only have to have the same

molecular weight as the IP3R’s (Fig. 2B), but also its association with IP3R’s would have to

be sufficiently strong to withstand the stringent washing conditions used during precipitation

on avidin-beads (see Methods). We conclude, therefore, that these biotinylated receptors are

localized to the plasma membrane and in all likelihood are found there as integral membrane

proteins. As already discussed in some detail there is considerable evidence in the literature

for the presence of IP3R's in the plasma membranes of various cell types but recent

observations concerning close associations between the plasma membrane and the

endoplasmic reticulum (19-22) have raised some questions about the interpretations of these

data. The present results, however, leave little doubt that IP3R's occur as integral plasma

membrane proteins.

Putney (4) has proposed that IP3R-3 may operate as a capacitative Ca2+ entry channel

in some cell types, and DeLisle et al (14) observed that IP3R-3, but not IP3R-1, was detected

at the plasma membrane of Xenopus laevis oocytes. But our results indicate that presence in

the plasma membrane is not restricted to IP3R-3. Indeed, in our experiments ~85% of the

IP3R's found in the plasma membrane of Jurkat cells and >40% of the IP3R's found in the

plasma membrane of rat parotid cells were of the IP3R-1 and IP3R-2 subtypes. In this regard,

it is also worth mentioning that the tissues used in several of the studies providing evidence

for plasma membrane IP3R's (see Introduction) show little or no expression of IP3R-3. It also

seems clear that IP3R-3 can function effectively in the traditional role of an IP3R, as an

intracellular IP3-gated Ca2+ channel, since this is the only IP3R subtype expressed in A431


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cells and since essentially normal IP3-induced Ca2+ release has been demonstrated in a

genetically engineered B cell mutant expressing only IP3R-3 (29). Interestingly, in contrast to

the results of DeLisle et al (14) with Xenopus oocytes, in these latter studies, mutant B cells

expressing both IP3R-1 and IP3R-3 did not show a significant increase in IP3-induced Ca2+

entry over cells expressing IP3R-1 alone (29).

The physiological role of plasma membrane IP3R's remains to be determined. As

mentioned above, a role as a capacitative Ca2+ entry channel distinct from trp has been

proposed (4). Alternatively, non-capacitative Ca2+ entry pathways have also been observed in

many cell types (30) and plasma membrane IP3R's have been suggested as possible candidates

for these (30-32). Additional work will be required to explore these possibilities.


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References

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Footnotes

The abbreviations used are: avidin-HRP, peroxidase-conjugated avidin; DTT,

dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; HB, homogenation buffer; HBSS-H,

Hanks' balanced salt solution buffered with Hepes; IP3R, inositol 1,4,5 trisphosphate receptor;

PBS, phosphate buffered saline; TPCK, tosyl-L-phenylalanine chloromethyl ketone; Tris,

Tris(hydroxymethyl)aminomethane.


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Figure Legends

Fig. 1. Characterization of anti-IP3R antibodies.

A, A microsomal preparation (see Methods) from rat lung (10 µg protein/lane) was analysed

by immunoblotting with ABsI (lane 1, I), ABsII (lane 2, II), or ABsIII (Lane 3, III) as

described in Methods. The positions of the prestained molecular weight markers are indicated

on the left-hand side of the panel.

B, Microsomal preparations from rat cerebellum, rat liver, and A431 cells were solubilized

and immunoprecipitated with ABsI (IP3R-1), ABsII (IP3R-2), or ABsIII (IP3R-3), respectively

(see Methods). The resulting immunoprecipitates were then analysed by immunoblotting with

ABsI (lane 1; I), ABsII (lane 2; II), and ABsIII (lane 3; III).


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Fig. 2. Biotinylation of IP3R-3 in intact A431 cells.

A, A microsomal preparation from A431 cells (10 µg/lane) was analysed by immunoblotting

with ABsI (lane 1, I), ABsII (lane 2, II), or ABsIII (Lane 3, III) as described in Methods.

B, The external surface of A431 cells was biotinylated with sulfo-NHS-biotin as described

in Methods. The resulting externally-biotinylated cell samples were then solubilized and

immunoprecipitated (IPP) with ABsIII in the presence (lanes 2 and 4) or absence (lanes 1 and

3) of the IP3R-3 C-terminal peptide against which ABsIII was raised (1 µg/ml; CT-peptide).

The resulting immunoprecipitates were then analysed by immunoblotting with either ABsIII

(lanes 1 and 2; III) or avidin-HRP (lanes 3 and 4; Avi) .

C, Biotinylated proteins were isolated from externally-biotinylated A431 cells or from non-

biotinylated controls by precipitation (PP) on avidin beads (Avi) as described in Methods.

The following samples were then analysed by immunoblotting with ABsIII: the original

extract from externally-biotinylated A431 cells (lane 1); the original extract from non-

biotinylated A431 cells (lane 2); the avidin-bound fraction from externally-biotinylated A431

cells when avidin precipitation was carried out as described in Methods (lane 3), or in the

presence of 5 µg/ml added free biotin (lane 4); the avidin-bound fraction from non-

biotinylated A431 cells (lane 5).


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Fig. 3.Analysis of biotinylated IP3R-3 and calnexin in sulfo-NHS-biotin-treated intact A431

cells and homogenates.

A and B, Biotinylated proteins were isolated from externally-biotinylated A431 cells on

avidin beads as described in Methods. Equivalent volumes of the following samples were

then analysed by immunoblotting with anti-calnexin antibody (CLN; panel A) or ABsIII (III;

panel B): the original extract from externally-biotinylated A431 cells (lane 1); the avidin-

unbound fraction (lane 2); and the avidin-bound fraction (lane 3). In addition, a sample three

times the volume of the avidin bound-sample in lane 3, was run in lane 4.

C and D, Biotinylated proteins were isolated from biotinylated-homogenates of A431 cells

on avidin beads as described in Methods. Equivalent volumes of the following samples were

then analysed by immunoblotting with anti-calnexin antibody (CLN; panel C) or ABsIII (III;

panel D): the original extract of the biotinylated homogenate (lane 1); the avidin-unbound

fraction (lane 2); and the avidin-bound fraction (lane 3).


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Fig. 4. Analysis of biotinylated IP3R's in sulfo-NHS-biotin-treated intact Jurkat cells.

A, A microsomal preparation from Jurkat cells (10 µg/lane) was analysed by

immunoblotting with ABsI (lane 1, I), ABsII (lane 2, II), or ABsIII (Lane 3, III) as described

in Methods.

B, Biotinylated proteins were isolated from externally-biotinylated Jurkat cells on avidin

beads as described in Methods. Equivalent volumes of the following samples were then

analysed by immunoblotting with ABsI (I), ABsII (II), ABsIII (III) or anti-calnexin antibody

(CLN): the original extract from externally-biotinylated Jurkat cells (lane 1); the avidin-

unbound fraction (lane 2); and the avidin-bound fraction (lane 3). In addition, a sample three

times the volume of the avidin bound-sample in lane 3, was run in lane 4.

Fig. 5. Analysis of biotinylated IP3R's in sulfo-NHS-biotin-treated intact rat parotid acini.

A, A microsomal preparation from dispersed rat parotid acini (10 µg/lane) was analysed by

immunoblotting with ABsI (lane 1, I), ABsII (lane 2, II), or ABsIII (Lane 3, III) as described

in Methods.

B, Biotinylated proteins were isolated from externally-biotinylated rat parotid cells and

analysed as in Fig. 4B.


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Table 1. Percentage of IP3 receptors on the plasma membrane.

Immunoreactivity of IP3R-1, IP3R-2 and IP3R-3 in experiments like those shown in Fig. 3-5

was quantified densitometrically. These values were then used to calculate the percentage of

each IP3 receptor subtype in the avidin-bound sample relative to its level in original extract.

Data shown are means ± S.E. (number of experiment). ND, not detectable.


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Akihiko Tanimura, Yosuke Tojyo and R. James Turnerintegral plasma membrane proteins

Evidence that type I, II, and III inositol 1,4,5 trisphosphate receptors can occur as

published online June 28, 2000J. Biol. Chem.

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