Distinct Functions of the Two Protein Tyrosine Phosphatase Domains of LAR (Leukocyte Common Antigen-Related) on Tyrosine Dephosphorylation of Insulin Receptor

Kazutake Tsujikawa, Naoto Kawakami, Yukiko Uchino, Tomoko Ichijo, Tatsuhiko Furukawa, Haruo Saito and Hiroshi Yamamoto

Department of Immunology (K.T., N.K., Y.U., T.I., H.Y.) Graduate School of Pharmaceutical Sciences Osaka University Osaka 565-0871, Japan
Department of Cancer Chemotherapy (T.F.) Institute for Cancer Research Kagoshima University, Faculty of Medicine Kagoshima 890-8520, Japan
Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology (H.S.) Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Protein tyrosine phosphorylation, which is an important mechanism for cellular signal transduction, is regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases) (1, 2). Whereas the biological functions and regulatory mechanisms of PTKs are well characterized, those of PTPases remain to be clarified. PTPases contain at least one conserved catalytic domain (known as the PTPase domain) that is composed of approximately 250 amino acids with a conserved signature motif ([I/V]HCXAGXXR[S/T]G) around the active site. The cysteine in the motif is the catalytic nucleophile that mediates the dephosphorylation reaction through the formation of a thio-phosphate bond (3). Mammalian PTPases that have been cloned to date are structurally divided into two subgroups: intracellular PTPases and transmembrane receptor-like PTPases (4, 5). All known intracellular PTPases possess only one PTPase domain. Many of them, however, also have accessory domains such as the Src homology 2 (SH2) domain, the ezrin-like domain, or the Pro/Glu/Ser/Thr-rich (PEST) domain. These accessory domains may regulate the activity, substrate specificity, or subcellular localization of the intracellular PTPases (6, 7).

In contrast, many receptor-like PTPases have two tandemly repeated cytoplasmic PTPase domains. The membrane-proximal and membrane-distal PTPase domains are called D1 (domain 1) and D2 (domain 2), respectively. Previous studies on the structure-function relationships of the receptor-like PTPases indicated that: 1) D1 has PTPase activity by itself; 2) the conserved cysteine residue in the signature motif of D1 is essential for the PTPase activity; 3) the three-dimensional structures of D1 and D2 are very similar; and 4) D2 has no or very little intrinsic catalytic activity (8, 9, 10, 11). These results suggested that although D2 is structurally very similar to D1, its function may be to regulate the catalytic activity or specificity of D1.

Extracellular domains of receptor-like PTPases are composed of various combinations of functional motifs such as the immunoglobulin-like (Ig) repeat, the fibronectin type III (FnIII) repeat, and the carbonic anhydrase-like domain, suggesting that the extracellular domain of receptor-like PTPases may modulate PTPase activity by binding to specific ligands. Specific ligands are, however, known for only a handful of receptor-like PTPases: PTPß binds contactin and other extracellular matrix molecules (12, 13, 14); PTP{kappa}, PTP{lambda}, and PTPµ are homotypic receptors (15, 16, 17); and laminin-nidogen complex binds to the fifth FnIII domain of human leukocyte common antigen-related (LAR) molecule (18). Although effects of ligand binding on PTPase activity are not known, it has been proposed that homodimerization of receptor-like PTPases CD45 and PTP{alpha} may inhibit their PTPase activities (19, 20). According to this model, PTPase activity might be regulated by modulation (either promotion or inhibition) of receptor dimerization induced by ligand binding. However, the crystal structures of LAR and PTPµ do not lend support to this model (11, 21).

Receptor-like PTPase LAR consists of non-covalently-bound subunits designated the E (extracellular) subunit and the P (phosphatase) subunit, generated by proteolytic cleavage of a single precursor protein between the eighth FnIII domain and a transmembrane segment (22, 23). The 150-kDa E subunit is composed of three Ig domains and eight FnIII domains, whereas the 85-kDa P subunit has a short extracellular domain, a transmembrane domain, and two tandemly repeated PTPase domains (D1 and D2) in the cytoplasm. The E and P subunits of LAR are connected by a noncovalent bond. There are several lines of circumstantial evidence that implicate LAR PTPase in insulin receptor signal transduction: 1) LAR is widely detected on a variety of insulin-sensitive tissues such as liver, muscle, and adipocytes (24, 25, 26); 2) LAR-deficient mice exhibit significantly lower levels of plasma glucose as well as a reduced rate of hepatic glucose production in the fasting state, and a significant resistance to insulin-stimulated glucose disposal and suppression of hepatic glucose output (27); 3) increased LAR PTPase activity toward the insulin receptor is detected in subcutaneous adipose tissue of obese subjects (28); 4) in in vitro experiments, the recombinant LAR PTPase catalytic domain dephosphorylates a regulatory phosphorylation site (Tyr-1150) in the insulin receptor ß-subunit (25); 5) in a rat hepatoma cell line in which the expression of the LAR protein is suppressed by antisense RNA, tyrosine phosphorylation of the insulin receptor is significantly enhanced after insulin stimulation (29); and 6) LAR binds to and dephosphorylates tyrosine-phosphorylated insulin receptor in Chinese hamster ovary cells that express human insulin receptor and LAR molecules (CHO-hIR/LAR) as well as in a rat hepatoma cell line (30, 31). These results suggest a negative regulatory role of LAR in insulin signaling, and the dysregulation of LAR PTPase activity might be a pathogenic factor in insulin-resistant diabetes.

The purpose of the current study is to clarify the functions of the two PTPase domains of LAR in the regulation of insulin receptor tyrosine dephosphorylation in vivo, using LAR mutants in which the catalytic cysteine residue in D1 or D2 has been changed to serine, or in which either D1 or D2 has been deleted. Using this approach, we showed that the LAR D1 is responsible for insulin receptor dephosphorylation, whereas the LAR D2 is mainly responsible for the recognition of the phosphorylated insulin receptor. Moreover, the association of extracellular domains of insulin receptor and LAR was supported by using a LAR deletion mutant without both PTPase domains. We found that LAR not only dephosphorylates insulin receptor but also serves as a substrate of insulin receptor tyrosine kinase. Thus, the extracellular domain and two PTPase domains of the receptor type PTPase LAR have distinct functional roles in insulin receptor signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activated Insulin Receptor Is Tyrosine Dephosphorylated by LAR
Previous in vitro studies of the LAR PTPase demonstrated that only the first PTPase domain (D1) has enzymatic activity, whereas the second PTPase domain (D2) has no detectable enzymatic activity but may modulate the activity of the PTPase D1. However, these results were obtained using artificial substrates such as phosphorylated oligopeptides. To investigate the functional distinctions of the two PTPase domains under more physiological conditions, we studied the in vivo regulation of one of its likely physiological substrates, namely the insulin receptor (IR), by LAR.

Initially, COS-7 cells were cotransfected with IR, together with the full-length LAR wild type (LAR WT), a LAR mutant that harbors the Cys-1522 to Ser substitution at the catalytic center of PTPase D1 (this mutation will be abbreviated as LAR D1CS), or the empty vector. The Cys-1522 to Ser mutation completely abolishes the in vitro LAR PTPase activity using artificial substrates. NP40 lysates were prepared from the transfected cells at various times after the stimulation with 100 nM insulin, and the tyrosine-phosphorylated proteins were detected by immunoblotting with antiphosphotyrosine monoclonal antibody (mAb) 4G10 (Fig. 1Go). A 95-kDa protein was prominently tyrosine phosphorylated 1 min after the insulin stimulation, and the tyrosine phosphorylation levels remained almost constant for 15 min after the insulin stimulation. When the same blot was reprobed with anti-IR ß-subunit antiserum, a 95-kDa protein band was detected (data not shown), indicating that the 95-kDa tyrosine-phosphorylated band is the insulin receptor.



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Figure 1. Tyrosine-Phosphorylated Proteins in Lysates of COS-7 Cells Transfected with Insulin Receptor and LAR WT or LAR D1CS

COS-7 cells were cotransfected with 1 µg of pSR{alpha}-IR (IR) and 5 µg of either pSP65-SR{alpha}2 vector (Mock), pSP65-SR{alpha}2-wild type LAR (LAR WT), or pSP65-SR{alpha}2-LAR D1CS (LAR D1CS), using the DEAE-dextran method. After incubation for 48 h, the transfected cells were serum starved for 16 h and then stimulated with 100 nM insulin for the indicated times. Cell lysates were electrophoresed on 12% SDS-PAGE and then transferred to a nitrocellulose membrane. The membrane was immunoblotted with antiphosphotyrosine mAb 4G10 ({alpha}pTyr). The relative intensity of tyrosine-phosphorylated insulin receptor ß-chain (indicated with an arrow) was determined by a densitometer.

 
The tyrosine-phosphorylated IR levels in LAR WT transfectants were markedly lower than those in LAR D1CS transfectants and mock transfectants at any time after the insulin stimulation. These results indicate that the IR ß-subunit is a possible substrate of the LAR PTPase and that the LAR PTPase D1CS mutant does not have catalytic activity in vivo.

In the LAR D1CS transfectants, several additional proteins were also tyrosine phosphorylated prominently, even in the absence of insulin stimulation. There are two possible explanations for the increased tyrosine phosphorylation of these proteins: 1) the IR tyrosine kinase remains in the active state, due to the inability of LAR D1CS to dephosphorylate, and continues to tyrosine phosphorylate other proteins; or 2) these phosphorylated proteins are also substrates of LAR. To determine which of these two explanations was correct, COS-7 cells were cotransfected with LAR D1CS and IR and stimulated with insulin for 1 min, and NP40 cell lysates were prepared. LAR in the lysates was immunoprecipitated with anti-LAR E subunit mAbs (a mixture of 11.1A, 75.3A, and 71.2E), and the precipitates were immunoblotted with the antiphosphotyrosine mAb 4G10. Cys-to-Ser mutants of several PTPases are known to tightly bind their specific tyrosine-phosphorylated substrates without dephosphorylating them (32, 33, 34, 35). By analogy, it might be expected that LAR D1CS would tightly bind the tyrosine-phosphorylated form of its physiological substrates. As shown in Fig. 2BGo, only three bands of phosphorylated proteins, at 200 kDa, 95 kDa, and 85 kDa, were detected in LAR D1CS immunoprecipitates. The 95-kDa protein is IR, because it was detected with anti-IR antiserum (Fig. 2AGo). The other two bands are the LAR precursor and the LAR P subunit (see below). These results favor the first explanation.



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Figure 2. Specific Binding of Insulin Receptor to LAR D1CS and Tyrosine Phosphorylation of LAR P Subunit

LAR D1CS was cotransfected with insulin receptor (IR) to COS-7 cells as described in Fig. 1Go. Cell lysates prepared 1 min after insulin stimulation were immunoprecipitated with anti-LAR E subunit mAbs, separated on 12% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was immunoblotted with (A) antiinsulin receptor ß-subunit antiserum ({alpha}IR), (B) antiphosphotyrosine mAb ({alpha}pTyr), or (C) anti-LAR P subunit mAb (YU1).

 
The LAR P Subunit Is Tyrosine Phosphorylated by Insulin Stimulation
LAR is synthesized as an approximately 200-kDa precursor protein that is cleaved by an endogenous protease into two subunits, the 150-kDa E subunit and the 85-kDa P subunit (23). The LAR P subunit contains the transmembrane segment and the entire cytoplasmic domain. Because the 200-kDa and 85-kDa tyrosine-phosphorylated proteins in LAR D1CS immunoprecipitated with anti-LAR E subunit mAbs (Fig. 2BGo) are about the same size as the LAR precursor and P subunit, respectively, we examined whether LAR is tyrosine phosphorylated. To this end, we generated a mAb (YU1) that recognizes the LAR P subunit by immunizing mice with a glutathione-S-transferase (GST)-LAR cytoplasmic domain fusion protein. The same filter as shown in Fig. 2BGo was reprobed with the anti-LAR P subunit mAb YU1. As shown in Fig. 2CGo, 200-kDa and 85-kDa protein bands, which correspond precisely to the tyrosine-phosphorylated proteins, were detected by YU1, indicating that the 200-kDa and 85-kDa tyrosine-phosphorylated proteins are the LAR precursor protein and the LAR P subunit, respectively.

LAR PTPase D2 Is Important for Recognition of the Insulin Receptor
To examine whether the LAR D1 and D2 play distinct roles in the LAR-IR interaction, we used three LAR mutants; namely LAR D1CS, LAR D2CS (Cys-1813 to Ser mutation in D2), and LAR D1D2CS (a mutant with both Cys-1522 to Ser in D1 and Cys-1813 to Ser in D2). These LAR mutants, or LAR WT, were individually transfected together with IR. Cell lysates were prepared 1 min after insulin stimulation, and LAR was immunoprecipitated with anti-LAR E subunit mAbs. Immunoblots with the antiphosphotyrosine mAb 4G10, the anti-LAR P subunit mAb YU1, and the anti-IR antiserum are shown in Fig. 3Go, and the relative intensities of bands detected with the antiphosphotyrosine mAb 4G10 were analyzed by densitometry (Fig. 4Go). Tyrosine-phosphorylated insulin receptor was efficiently coimmunoprecipitated with LAR D1CS, moderately coimmunoprecipitated with LAR WT and LAR D1D2CS, but only negligibly coimmunoprecipitated with LAR D2CS (Fig. 3AGo). Interestingly, the immunoblotting with anti-IR antiserum indicated that IR was present in the LAR WT and LAR D1CS immunoprecipitates at about the same level, but there was very little in the LAR D2CS or LAR D1D2CS immunoprecipitates (Fig. 3BGo), even though the levels of immunoprecipitated LAR (Fig. 3CGo) and the expression levels of insulin receptor (Fig. 3EGo) in these transfected cells were very similar. These results indicate that the cysteine residue in LAR D2 is important for the binding to the insulin receptor and that such D2-mediated binding is needed for efficient tyrosine dephosphorylation of the IR ß-subunit by the LAR D1 domain. Consistent with this interpretation, the total tyrosine phosphorylation level of IR in cell lysates of the LAR WT transfectant was significantly lower than those of LAR D1CS, D2CS, and D1D2CS (Figs. 1DGo and 4BGo), indicating that both D1 and D2 are important for IR dephosphorylation. Interestingly, the tyrosine phosphorylation levels of the LAR P subunit were significantly increased in the LAR D1CS and LAR D1D2CS transfectants compared with the LAR WT or LAR D2CS transfectant (Figs. 3AGo and 4AGo), suggesting that the phosphorylated tyrosine residue(s) of LAR P subunit is/are dephosphorylated mainly by the catalytic activity of LAR PTPase D1.



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Figure 3. Roles of LAR D1 and D2 on Tyrosine Dephosphorylation of, and Association with, Insulin Receptor

LAR WT, LAR D1CS, LAR D2CS, or LAR D1D2CS was cotransfected with insulin receptor (IR) to COS-7 cells as described in Fig. 1Go. Cell lysates prepared 1 min after insulin stimulation were immunoprecipitated (IP) with anti-LAR E subunit mAbs ({alpha}LAR E subunit), separated on 12% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was immunoblotted (Blot) with antiphosphotyrosine mAb ({alpha}pTyr) (A). The filter was stripped and reprobed with (B) antiinsulin receptor ß-subunit antiserum ({alpha}IR), and (C) anti-LAR P-subunit mAb (YU1), sequentially. Aliquots of total cell lysates were immunoblotted with (D) antiphosphotyrosine mAb ({alpha}pTyr) or (E) antiinsulin receptor ß-subunit antiserum ({alpha}IR).

 


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Figure 4. Densitometric Analysis of Tyrosine Phosphorylation Levels of Insulin Receptor and LAR P Subunit

The immunoblots with antiphosphotyrosine mAb shown in Fig. 3Go, panels A and D, were scanned by a densitometer, and the relative intensity of tyrosine phosphorylation levels of (A) insulin receptor associated with LAR (open bar) and LAR P-subunit (filled bar), and (B) insulin receptor in cell lysates were analyzed by an NIH image software.

 
To further clarify the distinct functions of LAR D1 and D2, we constructed three LAR deletion mutants, i.e. LAR {Delta}D1 and LAR {Delta}D2 (mutants lacking D1 and D2, respectively) and LAR {Delta}PTPase (a mutant lacking both D1 and D2). These LAR deletion mutants, or LAR CS, were individually transfected into COS-7 cells together with IR as described above. LAR was immunoprecipitated with anti-LAR E subunit mAbs from the cell lysates prepared 1 min after insulin stimulation. Figure 5AGo shows the immunoblot with the antiphosphotyrosine mAb PY20 conjugated with horseradish peroxidase. Tyrosine-phosphorylated IR was most prominently coimmunoprecipitated with LAR D1CS, was moderately so with LAR {Delta}PTPase and LAR {Delta}D1, and was only negligibly so with LAR {Delta}D2. When the same blot was reprobed with anti-LAR E subunit mAbs, almost the same intensities of 150-kDa bands were detected in transfectants of LAR D1CS, {Delta}D2, and {Delta}PTPase, but the intensity in LAR {Delta}D1 was much weaker (Fig. 5BGo). However, the obvious association of LAR {Delta}D1 with tyrosine-phosphorylated IR was detected in spite of its weak expression, indicating that LAR D2 plays an important role in the recognition of tyrosine-phosphorylated IR. Interestingly, the binding of tyrosine-phosphorylated IR to LAR {Delta}PTPase, which has no PTPase domains, was also detectable, suggesting the possibility of association between the extracellular domains of both LAR and IR. When the same blot was reprobed with anti-LAR P subunit mAb YU1, 85-kDa, 53-kDa, 51-kDa, and 22-kDa bands were detected in transfectants of LAR DCS, {Delta}D2, {Delta}D1, and {Delta}PTPase, respectively (Fig. 5CGo). These bands, with the exception of the 53-kDa band of LAR {Delta}D2, completely corresponded to the bands detected with an antiphosphotyrosine mAb in Fig. 5AGo, indicating that tyrosine residue(s) existing between the transmembrane domain and the PTPase domain 1 of LAR were tyrosine phosphorylated by IR. An in vitro PTPase assay indicated that LAR {Delta}D2 immunoprecipitated from COS-7 transfectants with anti-LAR E subunit mAbs had obvious PTPase activity, but LAR {Delta}D1 or LAR {Delta}PTPase immunoprecipitates had no activity (data not shown). Therefore, we supposed that since LAR {Delta}D2 could bind to tyrosine-phosphorylated IR and promptly dephosphorylated the IR and tyrosine-phosphorylated LAR, only a weak band of tyrosine-phosphorylated IR was detected.



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Figure 5. LAR {Delta}D1 and LAR {Delta}PTPase Associated with, and Tyrosine-Phosphorylated by, Insulin Receptor

LAR D1CS, LAR {Delta}D1, LAR {Delta}D2, or LAR {Delta}PTPase was cotransfected with insulin receptor (IR) to COS-7 cells as described in Fig. 1Go. Cell lysates prepared 1 min after insulin stimulation were immunoprecipitated (IP) with anti-LAR E subunit mAbs ({alpha}LAR E subunit), separated on 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was immunoblotted (Blot) with antiphosphotyrosine mAb conjugated to horseradish peroxidase ({alpha}pTyr) (A). The filter was stripped and reprobed with (B) anti-LAR E subunit mAbs ({alpha}LAR E subunit) and (C) anti-LAR P subunit mAb (YU1), sequentially.

 
The LAR P Subunit Is a Substrate for the Insulin Receptor Tyrosine Kinase
The LAR P subunit is tyrosine phosphorylated after insulin stimulation in transfected COS-7 cells. To examine whether LAR is phosphorylated by the insulin receptor tyrosine kinase, wild-type and kinase-inactive mutant insulin receptor constructs, in which Lys-1013 at the ATP binding site is mutated to Met, were used for transfection assays. Expression plasmids for the wild-type insulin receptor (IR WT) or the kinase-inactive mutant insulin receptor (IR MT) were cotransfected together with LAR D1CS. One minute after insulin stimulation, cell lysates were prepared and LAR was immunoprecipitated with anti-LAR E subunit mAbs, followed by immunoblotting with antiphosphotyrosine mAb 4G10. As shown in Fig. 6AGo, the LAR P subunit as well as the coprecipitated IR ß-subunit were tyrosine phosphorylated in cells transfected with the IR WT but not in those transfected with the IR MT. In these experiments, expression levels of both IR and LAR were comparable between the two transfected cells (data not shown). These results thus indicate that the LAR P subunit is a substrate of the insulin receptor tyrosine kinase. Moreover, when the same blot was reprobed with the anti-IR antiserum, IR was detected in both IR WT and IR MT transfectants (Fig. 6BGo), indicating that part of LAR-IR association is independent of the IR phosphorylation state.



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Figure 6. Tyrosine Phosphorylation of a LAR P Subunit by Insulin Receptor Tyrosine Kinase

LAR D1CS was cotransfected with an empty vector (Mock), IR WT, or IR MT to COS-7 cells as described in Fig. 1Go. Cell lysates prepared 1 min after insulin stimulation were immunoprecipitated (IP) with anti-LAR E subunit mAbs ({alpha}LAR E subunit), separated on 12% SDS-PAGE, and transferred to nitrocellulose. The membrane was immunoblotted with (A) antiphosphotyrosine mAb ({alpha}pTyr) or (B) antiinsulin receptor ß-subunit antiserum ({alpha}IR).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Most receptor-like PTPases such as LAR have two tandemly repeated PTPase domains in the cytoplasmic segment, although the precise function and the regulatory mechanism of each PTPase domain have not been clarified. Previous studies indicated that the membrane-proximal PTPase D1 of LAR has catalytic activity by itself, since the cysteine to serine-substituted mutation at the catalytic center of D1 completely eliminated the PTPase activity (9). In contrast, the membrane-distal PTPase D2 has no detectable enzymatic activity, even though its structure is strikingly similar to D1 (11). Removal of LAR PTPase D2, however, altered the specificity to artificial in vitro substrates, myelin basic protein, and the synthetic peptide Raytide, suggesting that the PTPase D2 might regulate substrate specificity. To clarify the precise function and the regulatory mechanism of each PTPase domain of LAR in vivo, however, studies using a physiological substrate of LAR are essential.

In this study, our criteria for a physiological substrate of LAR were as follows: 1) a LAR substrate is dephosphorylated more efficiently by LAR PTPase than other tyrosine-phosphorylated proteins, and 2) a tyrosine-phosphorylated substrate of LAR has a high affinity to LAR PTPase D1. We demonstrated preferential dephosphorylation of tyrosine-phosphorylated insulin receptor by LAR PTPase and a specific association of LAR D1CS mutant with the tyrosine-phosphorylated insulin receptor. Recently, Ahmad et al. (30, 31) also reported a physical association of rat LAR and the human insulin receptor overexpressed in CHO cells. The association seems to increase in accordance with the tyrosine phosphorylation of insulin receptor. These results indicate that the insulin receptor is a likely physiological substrate of LAR. Based on these findings, our present study has further clarified distinct functions of the two PTPase domains of LAR for the insulin receptor by various kinds of LAR mutants in vivo.

In cotransfection experiments, we found that LAR WT efficiently dephosphorylated the insulin receptor activated by insulin stimulation. On the other hand, the tyrosine-phosphorylated insulin receptor was coprecipitated with LAR D1CS, a substrate trapping mutant of LAR, indicating the tight association of these two molecules. These results support that LAR PTPase D1 is involved in a catalytic process, and that Cys-1522 located at the catalytic center of the PTPase D1 is critical for its catalytic activity in vitro (9) as well as in vivo. In the same way, it might be expected that if LAR PTPase D2 has a catalytic effect on the phosphorylated insulin receptor, both LAR D2CS and LAR D1D2CS mutants could efficiently trap the tyrosine-phosphorylated insulin receptor as well. However, neither LAR D2CS nor LAR D1D2CS showed a marked binding to the phosphorylated insulin receptor as was seen in LAR D1CS, suggesting that LAR PTPase D2 does not catalyze the dephosphorylation of the insulin receptor. Interestingly, the association of the insulin receptor to LAR constructs that have no mutation in the domain 2, such as LAR WT and LAR D1CS, is significantly weakened by the introduction of Cys to Ser-substituted mutation in LAR D2. In contrast, LAR {Delta}D1 that has only a wild-type PTPase domain 2 in the cytoplasm showed obvious binding to the phosphorylated insulin receptor. These results indicate that LAR PTPase D2 is important for the recognition of a substrate, the insulin receptor, and that Cys-1813 in the domain is essential for recognition in vivo.

Ahmad and Goldstein (31) have reported that LAR is associated with the insulin receptor and that the association is enhanced by insulin stimulation. We also confirmed the physical association between LAR and the insulin receptor in transfected COS-7 cells. However, LAR {Delta}PTPase was found to be associated with the insulin receptor in spite of the complete absence of LAR PTPase domains. These results indicated that not only the PTPase domains of LAR but also its extracellular domain are involved in the association with the insulin receptor.

LAR undergoes proteolytic processing when cells are grown to a high density or in response to increases in cytoplasmic Ca levels and protein kinase C activity (23). Aicher et al. (36) have reported that the processing of LAR is detected 5 min and 40 min after calcium ionophore stimulation in A431 and Hela cells, respectively. In our transfection assay of COS-7 cells, we could hardly detect the shed LAR in the culture media at 1 min, and we could detect it only slightly at 5 min after insulin stimulation (data not shown). Therefore, the processing of the LAR extracellular domain would not affect the interpretation of the results from immunoprecipitation assays with anti-LAR E subunit mAbs in this study.

LAR was tyrosine phosphorylated by tyrosine kinase activity of insulin receptor in response to insulin stimulation and rapidly autodephosphorylated in vivo. Some tyrosine phosphatases, such as PTP1C (37), PTP{alpha} (38), and PTPase 1B (39), are also known to be tyrosine phosphorylated by the insulin receptor tyrosine kinase. The phosphorylation of PTP1C increases its PTPase activity, and that of PTPase 1B is necessary for its interaction with insulin receptor. The tyrosine phosphorylation of PTP{alpha} leads to a reduction of the insulin receptor signaling (the activation of an endogenous kinases, such as Src, and the binding of Grb2) (40). The tyrosine phosphorylation sites in LAR, the functional significance of the phosphorylation, and the understanding of the change, if there is any, in the catalytic activity caused by the phosphorylation are unclear. Nonetheless, the phosphorylation and autodephosphorylation of LAR tyrosine residues are likely to play important functions in the regulatory mechanism of insulin-receptor signal transduction.

Based on the results of this study, we propose a new model of the tyrosine dephosphorylation mechanism of insulin receptor by LAR PTPase. LAR weakly associates with the insulin receptor at the steady state via the LAR extracellular domain. After insulin binding, insulin receptor tyrosine kinase induces autophosphorylation, leading to increased tyrosine kinase activity. The activated insulin receptor tyrosine kinase phosphorylates its substrates, including LAR. The tyrosine phosphorylation of LAR might induce the conformational change of the PTPases D1 and/or D2, resulting in a tighter association via LAR PTPase domain 2, and then dephosphorylating phosphotyrosine residues of the insulin receptor by the catalytic activity of the PTPase D1. A phosphorylated tyrosine residue(s) of LAR is also dephosphorylated by the catalytic activity of the PTPase D1 to return to the preactivation state.

Insulin receptor signaling is important for both cell growth and glucose metabolism. Molecular links between LAR and phosphorylated tyrosine residues of the insulin receptor are suggested. However, the elaborate regulatory mechanism by the two PTPase domains of LAR must still be clarified, perhaps using both the insulin receptor mutants with substitutions of tyrosine resides and various LAR mutants.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
The LAR expression vectors (WT, D1CS, D2CS, and D1D2CS) were constructed by inserting LAR and its Cys to Ser mutants described previously (9) into a modified version of the pcDL-SR{alpha}296 expression plasmid termed pSP65-SR{alpha}2 (41). pSP65-SR{alpha}2-LAR {Delta}PTPase (amino acids 1–1,343) and pSP65-SR{alpha}2-LAR {Delta}D2 (amino acids 1–1,590) deletion mutants were constructed by removing appropriate restriction fragments from pSP65-SR{alpha}2-LAR WT. A cDNA sequence corresponding to LAR PTPase domain 2 (amino acids 1,633–1,881) with appropriate restriction sites for subcloning were generated by PCR and inserted in-frame into the pSP65-SR{alpha}2-LAR {Delta}PTPase to construct LAR {Delta}D1. Their nucleotide sequences were confirmed by DNA sequencing. The numbering of LAR amino acid residues is in accordance with Streuli et al. (22). Human insulin receptor expression vectors, pSR{alpha}-human insulin receptor (pSR{alpha}-IR) and pSR{alpha}-IR K1030M, in which Lys-1030 at the ATP binding site of the insulin receptor is changed to Met, were kindly provided by Dr. Ebina.

Cell Culture and Transfections
COS-7 cells, obtained from the Human Science Research Resources Bank (Osaka, Japan), were grown at 37 C in a 5% CO2 atmosphere in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% heat-inactivated FCS (Life Technologies, Inc., Gaithersburg, MD) and 10 mg/ml kanamycin. Cells were cotransfected with 5 µg of pSP65-SR{alpha}2-LAR and 1 µg of pSR{alpha}-IR by the diethylaminoethyl (DEAE)-dextran method (42). After incubation for 48 h, the transfected cells were starved for 16 h in serum-free RPMI 1640 medium and then stimulated with 100 nM insulin for the indicated times. After washing with ice-cold PBS containing 1 mM sodium vanadate, 5 mM NaF, 5 mM sodium pyrophosphate, and 5 mM EDTA, the cells were lysed on ice in 1 ml of ice-cold lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris, pH 7.4, 5 mM EDTA, 10 mM iodoacetamide, 10 mM NaF, 10 mM sodium pyrophosphate, 0.4 mM sodium vanadate, 0.1 mM phenylarsine oxide, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine). The cell lysates were centrifuged in a microcentrifuge to remove insoluble materials before immunoprecipitation and immunoblotting.

Immunoprecipitation and Immunoblotting
Cell lysates were precleared with approximately 15 µg isotype-matched control mAbs (Transduction Laboratories, Inc., Lexington, KY) and 20 µl Gamma-bind (Amersham Pharmacia Biotech, Arlington Heights, IL) for more than 2 h at 4 C. For immunoprecipitation, cell lysates were incubated with 15 µg monoclonal anti-LAR mAbs (1:1 mixture of 11.1A and 75.3A) (23) for 1 h at 4 C, and then with 20 µl Gamma-bind for an additional 1 h at 4 C. After washing twice with 1 ml lysis buffer as described above and once with 1 ml PBS with 10 mM NaF, 10 mM sodium pyrophosphate, 0.4 mM sodium vanadate, and 0.1 mM phenylarsine oxide, immunoprecipitates were resolved by SDS-PAGE. Proteins in immunoprecipitates and cell lysates were transferred from a SDS-polyacrylamide gel to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH), blocked with 3% BSA in TBS-T (20 mM Tris-HCl pH 8.0, 137 mM NaCl, and 0.1% Tween 20) and incubated with primary mAbs at room temperature for 1 h, followed by washing three times with TBS-T. To detect antibody binding, horseradish peroxidase-conjugated antimouse IgG or horseradish peroxidase-conjugated antirabbit IgG (Transduction Laboratories, Inc.) diluted in TBS-T was incubated at room temperature for 1 h. After washing in TBS-T three times, bound horseradish peroxidase conjugates were visualized with enhanced chemiluminescent reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The antibodies used were antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), antiinsulin receptor ß-subunit (Transduction Laboratories, Inc.), anti-LAR E subunit mAbs (1:1:1 mixture of 75.3A, 11.1A, and 71.2E) (23), and anti-LAR P subunit mAb (YU1). For the blot of immunoprecipitates from lysates of COS-7 cells cotransfected with LAR deletion mutants and IR, we used antiphosphotyrosine mAb PY20 conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Preparation of Antihuman LAR P Subunit mAb (YU1)
The anti-LAR P subunit mAb YU1 was generated by immunization with a recombinant glutathione S-transferase fusion protein with the LAR P subunit beyond transmembrane segment expressed in a pGEX-2T vector (Amersham Pharmacia Biotech).


    ACKNOWLEDGMENTS
 
We thank Dr. Michel Streuli (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) for providing human LAR expression vectors and anti-LAR monoclonal antibodies, and Dr. Yousuke Ebina (the Department of Enzyme Genetics, the University of Tokushima, Tokushima, Japan) for human insulin receptor expression vectors. We also appreciate Dr. Pauline O’Gray (Dana-Farber Cancer Institute, Harvard Medical School) and Dr. Masato Kasuga (the Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan) for helpful advice.


    FOOTNOTES
 
Address requests for reprints to: Dr. Kazutake Tsujikawa, Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, 1–6 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: tujikawa{at}phs osaka-u.ac.jp.

This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, and a grant from Fuso Pharmaceutical Industries, Ltd.

Received for publication January 19, 2000. Revision received September 25, 2000. Accepted for publication October 13, 2000.


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 MATERIALS AND METHODS
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