Cloning and characterization of islet cell antigen-related protein-tyrosine phosphatase (PTP), a novel receptor-like PTP and autoantigen in insulin-dependent diabetes.

Cloning of the cDNA encoding a novel human protein- tyrosine phosphatase (PTP) called islet cell antigen-related PTP (IAR) predicts a receptor-like molecule with an extracellular domain of 614 amino acids containing a hydrophobic signal peptide, one potential N-glycosylation site, and an RGDS peptide which is a possible adhesive recognition sequence. The 376-amino acid intracellular region contains a single catalytic domain. Recombinant IAR polypeptide has phosphatase activity. Northern blot analysis shows tissue-specific expression of two IAR transcripts of 5.5 and 3.7 kilobases, which are most abundant in brain and pancreas. The IAR PTP is homologous in its intracellular region to IA-2, a putative PTP that is an insulin-dependent diabetes mellitus (IDDM) autoantigen. IAR is also reactive with IDDM patient sera. IAR and IA-2 may distinguish different populations of IDDM autoantibodies since they identify overlapping but nonidentical sets of IDDM patients. Thus IAR is likely to be an islet cell antigen useful in the preclinical screening of individuals for risk of IDDM.

The co-ordinated actions of protein tyrosine kinases and phosphatases (PTPs) 1 control much of the reversible protein phosphorylation central to eukaryotic cell proliferation and differentiation. As has been found for the protein tyrosine kinases, the ever expanding number and structural diversity of members of the PTP family suggest that these enzymes are critical and specific regulators of cellular processes (1,2). All PTPs possess one or two conserved catalytic domains. The highly conserved active site within each domain contains an essential cysteine residue that functions as a transient phosphate acceptor during the dephosphorylation reaction (3). This sequence conservation has permitted the PCR-based isolation of many novel PTPases. Nonreceptor PTPs with a single catalytic domain and receptor-like PTPs generally possessing two intracellular catalytic domains have been isolated. The regions flanking the catalytic domain of nonreceptor PTPs often contain structures responsible for subcellular localization and/or enzymatic regulation, for example N-terminal SH2 domains or C-terminal hydrophobic tails (reviewed in Ref. 4). The receptorlike PTPs consist of an intracellular region with the catalytic domains, a transmembrane region, and an extracellular region that often, but not always, contains structural motifs found in cell adhesion such as FN-III and Ig-like repeats. The receptor-like structure of many PTPs suggests that they act as transducers of extracellular signals and that appropriate intracellular signaling pathways are initiated by tyrosine dephosphorylation events.
Several PTPs have been implicated in disease processes. The pathogenic Yersinia bacteria, causing plague or other often fatal gastrointestinal disorders, contain an essential virulence determinant which is a PTP (5). The secreted PTP may act to abrogate the host response to bacterial infection (6). Mice with a defective gene encoding the nonreceptor SH-PTP1 (HCP, PTP-1C) exhibit multiple severe hematopoietic defects (7), which may be due to a lack of negative regulation of cytokine signaling. There is also evidence that some PTPs are potential oncogenes; overexpressed receptor-like PTP␣ is transforming, and elevated levels of PTP␣ message are present in 70% of colon carcinomas (8,9), and one isoform of the PTP cdc25 is transforming while another is overexpressed in 32% of human breast cancers (10).
Insulin-dependent diabetes mellitus (IDDM) is believed to result from the autoimmune-mediated destruction of pancreatic beta cells. Serum antibodies reactive with islet cell components can often be detected months or years before the disease is clinically apparent. These include the unidentified ICA antigen (11), insulin (12), glutamate decarboxylase (13), and 37and 40-kDa tryptic fragments of islet extracts (14). The use of single antibody markers in preclinical IDDM screening is limited by their comparatively low positive predictive value, but this has been improved by the use of combined markers (15). Identification of novel autoantigens that can be used in combination with existing markers is therefore an important priority. We report here the cloning and characterization of a human receptor-like PTP, termed IAR, which is reactive with IDDM sera. A study with a small group of IDDM patients suggests that IAR reactivity delineates a unique population of autoantibodies.

EXPERIMENTAL PROCEDURES
cDNA Isolation and Sequencing-Primers corresponding to conserved amino acid sequences (DYINA and VHCSAGV) of the catalytic domain of PTPases were designed (with EcoRI sites added) and used to amplify a human colon carcinoma cell (SW480) cDNA library (Clontech, HL3014b) using PCR. The primers had the sequences 5Ј-GGGAATTC-NGAYTAYATHAAYGC-3Ј and 5Ј-GGGAATTCACNCCNGCRCTRCA-RTGNAC-3Ј, where N is G, A, T, or C; Y is C or T; H is A, C, or T; and R is A or G. The major amplified fragment of ϳ460 bp was subcloned into the pGEM-T vector (Promega). The 3Ј RACE and 5Ј RACE used the Marathon cDNA Amplification Kit (Clontech), and cDNA was reversetranscribed from human brain poly(A) ϩ mRNA (Clontech) using the cDNA synthesis primer provided with the kit (3Ј RACE) or random primers or an IAR-specific primer (P5) with the sequence 5Ј-CGTGTG-GGCCACATAGGTCAGGATGCTCTCGGAGAA-3Ј (5Ј RACE). The PCR amplification was carried out using either a forward or reverse primer corresponding to the ligated adaptor in combination with an IAR-specific forward primer, 5Ј-CCTGCCTCCTCAGGCGGAGCAAGA-3Ј (3Ј RACE) or an IAR-specific reverse primer (P11), 5Ј-TGGCGAGCACGT-CTGAGGCTG-3Ј (5Ј RACE). The amplified fragments were cloned into pGEM-T (Promega) and sequenced along both strands.
Analysis of Signal Peptide Function-The cDNA encoding the fulllength IAR protein was modified by replacing the 5Ј-untranslated region with the sequence CCACC and then cloned into pBluescript SK(ϩ) (Stratagene). In vitro transcription and translation of this or the B11 cDNA were carried out according to the manufacturer's protocol using the TNT Coupled Reticulocyte Lysate Systems (Promega) in the presence of [ 35 S]methionine and with or without the addition of canine microsomal membranes (Promega). After translation, the products were treated with or without proteinase K (Boehringer Mannheim) at a final concentration of 0.01 mg/ml on ice for 7.5 min, in the presence or absence of 0.1% Triton X-100. The reaction products were analyzed by 10% SDS-PAGE and autoradiography.
Expression, Purification, and Assay of Recombinant IAR-The primers P1 and P2, 5Ј-GGGCTCGAGTCTAGACAGGCTGAAGGAGAAGCT-CTC-3Ј (with added XhoI and XbaI sites) and 5Ј-GGGGAATTCCATG-GTTATAATAGAAGACACACA-3Ј (with added EcoRI and NcoI sites), were used in a PCR with the 3-7 clone as template to amplify the intracellular region of IAR encoding amino acids 646-1015. The amplified fragment was cloned into the XhoI and EcoRI sites of pGEX-3C (17). Sequencing of the insert confirmed that no mutations had resulted from the PCR. Mutant IAR (C945S) was generated by PCR using overlapping forward and reverse primers corresponding to the desired mutation and surrounding sequence encoding IIVHSSDGA; 5Ј-ATAATTGTTCATTCCAGTGACGGTGCA-3Ј and 5Ј-TGCACCGTCAC-TGGAATGAACAATTAT-3Ј. Each primer was used in combination with one of the primers P1 or P2 (used to amplify the intracellular region of IAR) to generate two overlapping DNA fragments corresponding to nucleotides 1993-2904 and 2878 -3197 of IAR. These fragments were mixed and used as template for a PCR reaction with primers P1 and P2. The amplified fragment was cloned into pGEX-3C and verified by sequencing. Soluble GST-IAR fusion proteins were produced after isopropyl-1-thio-␤-D-galactopyranoside induction (0.15 mM) of Escherichia coli (DH5␣FЈ) transformed with the above plasmids. The GST-IAR was purified from bacterial lysates (17), and the IAR cleaved from GST using 3C protease (18). Purified IAR was quantitated by SDS-PAGE followed by densitometric scanning alongside known amounts of protein standards. Purified IAR was assayed as described (19) at 30°C in reactions with 50 mM sodium acetate (pH 4.5), 0.5 mg/ml BSA, 0.5 mM dithiothreitol, 2 or 5 mM p-NPP, plus or minus 1 mM Na 3 VO 4 , and plus or minus IAR or IAR(C945S) as indicated.
Patients and Sera-Sera were obtained from healthy blood bank controls (n ϭ 10) and from recent-onset IDDM patients (n ϭ 20, age 4.0 -15.7 years) prior to commencement of insulin therapy. ICA were measured by indirect immunofluorescence on frozen sections of human pancreas (20), and GAD antibodies were measured by enzymatic immunoprecipitation (21).
In Vitro Translation and Immunoprecipitation Tests with Patient Sera-cDNAs encoding amino acids 646-1015 of IAR (representing the intracellular region) or full-length IA-2 protein (16) (a gift from E. Bonifacio) were subcloned into the pGEM-3 vector, under the control of the SP-6 promoter. Radiolabeled IAR and IA-2 proteins were synthesized by in vitro translation and transcription in the presence of [ 35 S]methionine, using a reticulocyte lysate system (Promega TNT Kit). For immunoprecipitations, 1 l of lysate containing labeled IAR or IA-2 in 100 l of TBST-BSA (50 mM Tris-HCl, 150 mM NaCl, 1% Tween-20, 0.1% BSA) was incubated overnight at 4°C with 2 l of serum that had been prebound to 10 l (packed bead volume) of protein A-Sepharose (Pharmacia Biotech, Inc.). Beads were washed once with TBST-BSA, once with high salt wash buffer (0.5% Triton X-100, 200 mM NaCl, 50 mM NaH 2 PO 4 ), twice in PBS plus 0.5% Triton X-100, and once in 0.5% Triton X-100, 4.17 mM Tris, 192 mM glycine (pH 7.4). Precipitated proteins were resolved by 10% SDS-PAGE. Gels were dried and exposed to a phosphorimager, and bands were quantified by densitometry after subtraction of local background.

RESULTS
Identification and cDNA Cloning of IAR PTP-To search for novel PTPases, degenerate primers to conserved PTPase sequences were used in a PCR with human colon carcinoma cell cDNA library as template. The major ϳ460-bp amplified fragment was subcloned, and 94 clones were sequenced. The majority of inserts were found to be LAR (41 clones) and PTP␣ (23 clones) sequences. Another clone, r75, had 80% identity to a 274-bp region of IA-2 (ICA-512), a putative PTPase with receptor-like structure (16,22), and 96% identity in a region of overlap with a 386-bp brain EST03250 (23). Probing of human multiple tissue Northern blots with r75 detected high levels of transcript expression in brain and pancreas (data not shown, but see below).
r75 was used as probe to screen a human pancreas cDNA library. Several positive clones were isolated and identified as IA-2 cDNA by sequencing. Other clones encoded a distinct cDNA that contained r75 sequence. The largest of these, C3, was sequenced on both strands and found to contain an open reading frame of about 2.4 kb (Fig. 1). The C3 clone contained a coding sequence missing initiation and termination signals and was significantly smaller than the transcripts detected by Northern blotting with r75, suggesting that it was incomplete at both the 5Ј and 3Ј ends. To obtain the 3Ј end of the clone, 3Ј RACE was used on cDNA reverse-transcribed from human brain mRNA. Two different clones were obtained (Fig. 1). Clone 3-7 had a 5Ј region of identical overlap with C3 and additional sequence containing a stop codon and polyadenylation signal. Clone 3-10 was identical to 3-7 from the 5Ј end up to the 3-7 polyadenylation site, but was followed by approximately 2 kb of additional untranslated sequence. To obtain the 5Ј end of the cDNA, the pancreas library was rescreened with a 300-bp probe close to the 5Ј end of C3. No IA-2 clones were found. Of the positive clones, clone B11 appeared to have the longest 5Ј extension to C3 and was selected for further sequencing along both strands. The 3Ј end of B11 was identical to C3 sequence over a length of about 2 kb, but the 5Ј ends of B11 and C3 were different (Fig. 1). B11 had a unique 5Ј sequence of 412 bp, followed by a 54-bp sequence that is found in the opposite Both strands of clones B11, C3, and 3-7 were sequenced in their entirety. The angled 5Ј piece of C3 is not identical to the corresponding region of B11 and likely represents a cDNA library artifact, as does the boxed region (54 bp) of C3 that is a sequence found in the opposite orientation in the corresponding region of B11. orientation in C3. The sequence of C3 found 5Ј to this inverted region was shorter and different from that of B11. 5Ј RACE was used to determine which clone contained the correct 5Ј sequence and to try to obtain additional 5Ј sequence to the open reading frames of B11 and C3. Template cDNA was prepared from human brain poly(A) ϩ mRNA using random primers or a primer (P5) corresponding to shared B11/C3 sequence located about 170 bp 3Ј to the invert. The 5Ј RACE used a primer to the ligated adaptor sequence at the ends of the cDNA and a primer (P11) to a region of shared B11/C3 sequence located about 90 bp 3Ј to the invert. Cloning and sequencing of the 5Ј RACE PCR products from both types of template cDNA showed that all had B11 sequence. Similarly, PCR amplification of the pancreas cDNA library using a primer to gt11 and either P5 or P11 primers gave DNA fragments that all corresponded to the unique B11 sequence. Thus, the B11 sequence is correct, and the 5Ј end of the C3 clone is likely a library artifact. No PCR products were found with a 5Ј extension of the B11 sequence, and no 5Ј-extended clones were identified by further screening of human brain and pancreas cDNA libraries.
The complete cDNA is predicted to encode a receptor-like PTP of 1015 amino acids. This includes a 614-amino acid extracellular region, a 25-residue hydrophobic transmembrane segment, and a 376-amino acid intracellular region containing a single PTP catalytic domain (Fig. 2). This protein has about 43% overall sequence identity to the putative PTP IA-2 (16, 22), a protein identified as an islet cell autoantigen in IDDM, and another PTP-like molecule called PTPLP (24). We have thus named the novel PTP as islet cell antigen-related PTP or IAR PTP. An alignment of IAR and IA-2 amino acid sequences is shown in Fig. 3. The intracellular domain of IAR is 73% identical to that of IA-2; however, the identity falls to 24% in the extracellular regions of these proteins. The IAR extracellular region does not contain FN-III or Ig-like repeats but has the adhesion recognition peptide sequence RDGS (amino acids 372-375). There is one potential site for N-linked glycosylation. The catalytic domain of IAR has conserved sequences typical of other PTPases, although the active site is unusual in having an aspartate residue at position 947 (IVHCSDGAGRTG) in place of a conserved alanine residue (Fig. 2).
Although no in-frame stop codons were found 5Ј to the first ATG codon in the B11 sequence, we propose that this is the initiation codon for the following reasons. 1) A purine (G) in the Ϫ3 position conforms with the Kozak rules of initiation (25). 2) This methionine is followed by a 20-residue hydrophobic sequence that has the features of a signal sequence (26). To test for signal sequence function, a cDNA encoding the predicted full-length protein was transcribed and translated in vitro in the presence of [ 35 S]methionine and in the presence or absence of microsomal membranes. The protein synthesized in the absence of microsomal membranes (Fig. 4, lane 1) was completely digested upon subsequent treatment with proteinase K (Fig. 4, lane 2), whereas the protein synthesized in the presence of microsomal membranes (Fig. 4, lane 3) was reduced in size but partially protected from proteinase K digestion (Fig. 4, lane 4). The inclusion of Triton X-100 in the protease digestion resulted in no labeled protein remaining after proteinase K treatment, confirming that membrane integrity was necessary for protection from digestion (Fig. 4, lane 5). The same procedures were carried out with the shorter B11 cDNA (encoding a truncated IAR protein lacking the C-terminal half of the intracellular region). As expected, a smaller product was synthesized (Fig. 4,  lane 6), but a protected fragment was observed after proteinase K treatment that was the same size as that resulting from digestion of the full-length IAR protein (Fig. 4, lane 7), indicating that this is likely the extracellular region of IAR common to the products of both cDNAs. Together, these results suggest that the IAR protein is inserted into the membranes such that the extracellular region of IAR is in the proteinase K-inaccessible interior of the membrane vesicles and that the signal sequence is thus functional in directing the transmembrane insertion of IAR.
Expression of IAR-In view of the homology of IAR and IA-2, nonhomologous fragments of these cDNAs were selected to use as specific probes on Northern blots (Fig. 5). The two probes recognize distinct transcripts in terms of size and tissue specificity. The IA-2 probe detects a single transcript of about 3.9 kb, in accord with the results of Lan et al. (16). The highest expression of IA-2 is detected in brain, followed by spinal cord and pancreas, with low levels expressed in small intestine and adrenal gland. The IAR probe detects two transcripts of about 3.7 and 5.5 kb, with the highest expression in pancreas and brain, followed by trachea, prostate, stomach, and spinal cord with low levels detectable in small intestine and adrenal gland. It is possible that the 3.7-kb transcript has a 3Ј-untranslated sequence corresponding to that of the shorter 3-7 clone, whereas the 5.5-kb transcript represents an alternative 3Јuntranslated sequence corresponding to that of the longer 3-10 clone.
Catalytic Activity of IAR-The intracellular region of IAR (amino acids 646-1015) was expressed as a GST-fusion protein and purified following 3C protease cleavage from GST. The IAR migrated on SDS-PAGE as a major protein band of about 41 kDa (Fig. 6A, lane 2), in accord with the predicted size of 41.7 kDa. The purified IAR possessed low but detectable phosphatase activity toward p-NPP. The IAR is active over a narrow pH range, with optimal activity at pH 4.5, and is essentially inactive at pH 5.5 (Fig. 6B). IAR catalyzes the time-dependent dephosphorylation of p-NPP (Fig. 6C) with a specific activity of 21 nmol/min/mg. Like many PTPs, IAR activity is sensitive to inhibition by vanadate, and 1 mM sodium orthovanadate completely abolished activity (Fig. 6C). Site-directed mutagenesis of the essential cysteine residue (to a serine) in the active site resulted in the expression of an IAR intracellular region polypeptide which did not catalyze p-NPP hydrolysis (Fig. 6C), indicating that the phosphatase activity measured with the wild type polypeptide was unlikely to be due to contaminants in the protein preparation. IAR dephosphorylated phosphotyrosyl casein in a time-dependent manner, although with extremely low specific activity (2-3 pmol/min/mg), and about equivalent optimal activities were observed at pH 5.5 in sodium acetate buffer or at pH 6.5 in Mes buffer (data not shown). Phosphoseryl casein was not detectably dephosphorylated by IAR.
Reactivity of IAR with IDDM Patient Sera-Sera from 10 healthy blood bank control and 20 recent-onset IDDM subjects (ICA, GAD antibodies, age, and sex of subjects are shown in Table I)  Following translation, the reactions were treated with or without proteinase K (Prot K) or Triton X-100 (Tx-100) as indicated and analyzed by SDS-PAGE and autoradiography. The positions of molecular size markers (kDa) are indicated. At the top of the figure is a schematic diagram showing the full-length (FL) IAR protein (amino acids 1-1015) and the truncated protein product (amino acids 1-820) of the B11 cDNA. Numbering refers to amino acid positions; SP denotes the signal sequence, and TM denotes the transmembrane region. IDDM and control sera are shown in Fig. 7A. The immunoprecipitated 41-kDa band (corresponding in size to the recombinant IAR intracellular region polypeptide purified from bacteria, see above) was quantitated using a phosphorimager. Analysis of a panel of sera showed that IAR 41-kDa band densities were greater than the mean ϩ 2 (S.D.) of the 10 control sera (1818 density units) in 11 of the 20 (55%) recentonset IDDM sera (Table I). There was no significant correlation, in this small number of subjects, between IAR and ICA, GAD antibodies, or age. The IAR reactivity of the control and IDDM subjects is shown graphically in Fig. 7B. Sera from 9 of the 11 IAR-reactive patients also immunoprecipitated IA-2, some reacting more strongly with IAR than IA-2 and the inverse seen with others (Table I). Of interest is the finding that 3 IDDM subjects had antibodies that immunoprecipitated IAR or IA-2, but not both (Table I, subjects 4, 13, 20). Thus, IAR and IA-2 can distinguish different autoantibody populations in a subset of IDDM patients. DISCUSSION IAR is a member of a related group of receptor-like PTPs that includes IA-2 and PTPLP (16,22,24). These three molecules share sequence homology, have a common structure, and exhibit similar patterns of tissue-specific expression. Overall, IAR has 43.1% amino acid identity with human IA-2, and 42.6% identity with rat PTPLP, whereas IA-2 and PTPLP, with about 82% identity, are more closely related to one another.
The amino acid sequence of IAR is most distinct from that of IA-2 and PTPLP in its extracellular region, with 24.4 and 22.9% identity, respectively. However, all three PTPs are structurally alike in that they lack the FN-III and Ig-like repeats found in numerous other receptor-like PTPs and possess few sites for N-glycosylation. The sole feature of IAR suggestive of adhesive interaction is the RGDS peptide found at positions 372-375. The extracellular region of IA-2 also has this peptide motif, and it is present in PTPLP as RGDT. First recognized in fibronectin (27,28), this sequence can mediate cell attachment and binding to integrin receptors (29). The intracellular region of IAR is more closely related to those of IA-2 (73.4% identity) and PTPLP (74.4% identity). All have a single catalytic domain, within which the active site has an aspartate residue substituted for a conserved alanine (VHCSD). Alteration of this alanine residue is not observed with other receptor-like PTPs, with the exception of an identical substitution in the second (membrane distal) catalytic domain of CD45 (30). No phosphatase activity of IA-2 or PTPLP has been reported, and this has been suggested to be due to this alteration (22,24,31). However, IAR does have phosphatase activity, albeit low relative to that of many other PTPs. The low specific activity of IAR toward p-NPP may reflect a narrow substrate specificity of this phosphatase. Several nonreceptor PTPs have substitutions at the same position in the active site, for example bVH1 and cdc25 have a histidine residue in this position, whereas human VHR has a glutamic acid residue (32)(33)(34). These substitutions do not preclude catalysis, since all are active PTPs.
IAR is reactive with IDDM patient sera, identifying an overlapping set of subjects as does IA-2. Antibodies (termed ICA or islet cell antibodies) in the sera of IDDM patients react with islet cells in frozen sections of pancreas (11), and the nature of the reactive antigen(s) has long been sought. Autoantibodies recognize a 64-kDa polypeptide in detergent extracts of islet cells that was identified as glutamic acid decarboxylase (GAD) (13). Tryptic digestion of islet cell lysates reveals 50-, 37-, and 40-kDa IDDM-reactive antigens, of which only the 50-kDa form is reactive with anti-GAD antibody (14,35). Anti-GAD antibodies do not strictly correlate with IDDM, being found in patients with other autoimmune diseases (36). Serum antibodies to the 37/40-kDa antigens are strongly correlated with the development of IDDM in ICA-positive subjects, whether these be first degree relatives (15) or non-diabetic twins of IDDM patients (37), patients with polyendocrine autoimmunity (36), or selected by screening from the general population of schoolchildren (38). Recent efforts to identify the 37/40-kDa antigens have found that IA-2 polypeptide can block precipitation of the 40-kDa fragment by antibodies from non-diabetic relatives of IDDM patients, and partially block precipitation of the 37-kDa fragment, indicating that the antibodies can distinguish between what appear to be two related fragments (39,40). Furthermore, trypsin treatment of IA-2 generates a 40-kDa, but not a 37-kDa, fragment (40). This suggests that the 40-kDa fragment is IA-2, and the 37-kDa fragment is derived from a distinct but related protein, likely also to be a PTP. The recognition of IAR by IDDM antibodies and the identification of an overlapping yet distinct set of patients from IA-2, the sequence homology between IA-2 and IAR, particularly in the intracellular region that appears to be the antigenic region, and the expression of IAR in pancreas, all support the possibility that IAR is the precursor of the islet 37-kDa antigenic fragment.
The availability of recombinant antigenic polypeptides will greatly enhance the ease of screening general or IDDM-risk populations, and the sensitivity of such screens will be improved by employing more than one antigen. The IA-2 and IAR PTPs may serve such a purpose. Also of interest is the investigation of the cellular role of these PTPs in the pancreas and, if any, in IDDM development. FIG. 7. Reactivity of IAR with IDDM sera. A, immunoprecipitation of IAR with sera from control and recent-onset IDDM subjects. In vitro translated [ 35 S]methionine-labeled IAR was incubated with sera (prebound to protein A-Sepharose) from control (subjects 5 and 6 in Table I) (lanes 1 and 2) and IDDM subjects (subjects 12, 3, and 18 in Table I) (lanes [3][4][5]. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The 41-and 32-kDa bands (arrowed) are IAR and a likely IAR proteolytic product, respectively. B, graphical depiction of control and IDDM subject sera reactivity with IAR. The exact values of the data presented here and further subject information are given in Table I. Experiments were carried out as in A, and the immunoprecipitated IAR was resolved by SDS-PAGE. The signal from the 41-kDa IAR band was quantitated (in arbitrary density units) using a phosphorimager.
subjects; Peter Colman for serum samples; and Vijay Bhandari, Y. H. Tan, and Wanjin Hong for suggestions and critical reading of the manuscript.