©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternatively Spliced Juxtamembrane Domain of a Tyrosine Kinase Receptor Is a Multifunctional Regulatory Site
DELETION ALTERS CELLULAR TYROSINE PHOSPHORYLATION PATTERN AND FACILITATES BINDING OF PHOSPHATIDYLINOSITOL-3-OH KINASE TO THE HEPATOCYTE GROWTH FACTOR RECEPTOR (*)

(Received for publication, October 19, 1994; and in revised form, November 9, 1994)

Chong-Chou Lee (§) Kenneth M. Yamada (¶)

From the Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892-4370

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The hepatocyte growth factor (HGF) receptor is a tyrosine kinase receptor that mediates signal transduction upon ligand stimulation. This receptor is present in mouse tissues as two major isoforms differing by a 47-amino acid segment in the juxtamembrane domain, an alternatively spliced cytoplasmic region adjacent to the transmembrane domain of the receptor. We report here that the juxtamembrane domain of the receptor is involved in the regulation of downstream signal transduction. The two receptor isoforms were transiently expressed in COS-7 cells. Both exogenous receptors underwent autophosphorylation and subsequently stimulated a set of protein tyrosine phosphorylations that were not present in control cells. Comparisons of phosphotyrosine profiles of transfected cell lysates induced by receptor isoforms demonstrated that at least three phosphorylated proteins of 62, 35, and 30 kDa were differentially induced by the receptor isoforms, suggesting that the juxtamembrane domain of a kinase receptor can play a role in selective signal transduction. Furthermore, the p85 subunit of phosphatidylinositol-3-OH kinase (PI(3) kinase) co-precipitated with the small isoform of the HGF receptor, and this association was dramatically inhibited by treatment with 12-O-tetradecanoylphorbol-13-acetate. Since removal of the juxtamembrane domain facilitates the binding of p85 to the receptor, it is likely that the juxtamembrane region plays a role in negative regulation of the binding of PI(3) kinase to the HGF receptor. Our study establishes novel molecular sequelae of alternative splicing of an intracellular domain of the HGF receptor.


INTRODUCTION

The hepatocyte growth factor (HGF) (^1)receptor/c-met is a transmembrane protein that is a member of the tyrosine kinase receptor superfamily(1, 2) . The ligand for this tyrosine kinase receptor was identified as HGF, also known as scatter factor(3) . This factor has pleiotropic functions. It is a mitogen(4) , a morphogenic factor(5) , and an angiogenic factor(6) . Therefore, the HGF receptor is believed to be involved in the regulation of cell proliferation, migration, and differentiation. Upon HGF stimulation, the HGF receptor exhibits tyrosine kinase activity, including receptor autophosphorylation, and it activates a cascade of tyrosine phosphorylation involving a series of signal-transducing proteins. Following autophosphorylation, the HGF receptor associates with PI(3) kinase in vivo and in vitro(7, 8) . PI(3) kinase is a lipid kinase that phosphorylates phosphatidylinositols at the 3` position of inositol upon growth factor stimulation. The products generated by PI(3) kinase have been implicated in the regulation of cell growth. The p85 subunit of PI(3) kinase binds to tyrosine-phosphorylated sites of the HGF receptor through an SH2 domain (8) . The regulation of PI(3) kinase activity in response to growth factors can also be mediated by an intermediary cytoplasmic protein. Insulin receptor substrate-1, for instance, has multiple sequence motifs for PI3 kinase binding and is highly phosphorylated upon insulin stimulation(9) .

Besides PI(3) kinase activation, HGF/scatter factor can also stimulate the Ras-guanine nucleotide exchanger, which promotes the GTP-bound active state of Ras protein(10) . The Ras pathway is essential for the scattering effect mediated by HGF receptor(11) . Recently, Ras protein has been demonstrated to interact directly with PI(3) kinase in a GTP-dependent manner(12) . Since PI(3) kinase is a key enzyme in the intracellular growth signaling pathway, inhibitors have been developed as antiproliferative agents(13) . However, it is not clear whether the activation of PI(3) kinase can be differentially regulated by different forms of a tyrosine kinase receptor.

We have previously reported that a novel type of structural variant of the tyrosine kinase HGF receptor exists in mouse tissues(14) . This new isoform transcript, which is shorter by 141 base pairs, occurs through mRNA alternative splicing and results in a deletion of 47 amino acids in the juxtamembrane domain of the cytoplasmic domain of the receptor. The juxtamembrane domain appears to be involved in the negative regulation of kinase activity(15) , mitogenesis, and transforming activity(16) . These studies seem to suggest that this juxtamembrane domain may contain crucial determinants that could direct specificity of the signaling pathway of this kinase receptor.

The purpose of this study was to investigate the role of the juxtamembrane domain of the HGF receptor in the regulation of downstream signaling pathways by using HGF receptor isoform cDNA constructs. We report that the juxtamembrane domain is involved in selective tyrosine phosphorylation of three unidentified proteins. It is also a negative controlling element for the association of PI(3) kinase with the HGF receptor, since removal of the juxtamembrane region greatly facilitates binding of the p85 subunit of PI(3) kinase to the HGF receptor. Our data suggest a novel mechanism by which the HGF receptor may greatly reinforce its physical association with PI(3) kinase, promoting a specific signal transduction pathway among c-met-associated signaling cascades by using receptor isoforms that differ in a cytoplasmic juxtamembrane domain.


MATERIALS AND METHODS

Construction of HGF Receptor cDNA Expression Vector

Amplification of cDNA fragments corresponding to the mouse HGF receptor isoforms was carried out using the reverse transcription-polymerase chain reaction. Total RNA preparations from adult mouse kidney tissue were isolated using the guanidinium thiocyanate/phenol-based single-step method(17) . Poly(A) RNA was selected by oligo(dT)-cellulose chromatography as described (18) . To synthesize a DNA fragment encoding the 5`-half of the mouse HGF receptor, 1 µg of mRNA was hybridized to an antisense strand primer (m02: 5`-CGT CAT AGC GAA CTA ATT C-3` corresponding to nucleotides 2893-2911). The first strand cDNA was synthesized by reverse transcriptase (Perkin Elmer) at 42 °C. The sense strand primer used for polymerase chain reaction was m01 (5`-GAT CAACTC CTC ACA ATG AAG GCT C-3` = nucleotides -15 to 10), extended by a NotI restriction site. Another pair of oligonucleotides was utilized to synthesize the DNA fragment corresponding to the 3`-half of the HGF receptor: antisense and sense strand oligonucleotides m04 (5`-TGG GCC AGA ACT GTT TCT TGG A-3` = nucleotides 4166-4187) and m03 (5`-ATT TCT CCG AGG TAC GGC CC-3` = nucleotides 1981-2000), respectively. A NotI site was also included at the 3`-end of the molecule. The reaction was cycled 25 times, with denaturation for 1 min at 94 °C, annealing for 3 min at 60 °C, and extension for 3 min at 72 °C. The amplified polymerase chain reaction product was then fractionated on a 1% agarose gel. The bands of predicted sizes were excised, extracted, and cloned in the TA cloning vector (Invitrogen). The full-length cDNA for mouse HGF receptor was constructed by ligating two restriction fragments simultaneously into a pRBK plasmid vector (Invitrogen): a 2.9-kilobase NotI-MunI-restricted fragment that was synthesized by primers m01 and m02 and a 2.2-kilobase NotI-MunI-restricted fragment by primers m03 and m04. The 2.2-kilobase NotI/MunI fragment is a mixture of two DNA species differing by 144 base pairs. The sequences of the mouse HGF receptor cDNAs were determined by DNA sequence analysis using Sequenase kits (U. S. Biochemical Corp.).

Cell Culture and Transfection

COS-7 cells were grown overnight to 80-90% confluency in 100 times 20-mm dishes in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. A mixture of 6 µg of plasmid DNA and 36 µl of LipofectAmine (Life Technologies, Inc.) was incubated in 1.2 ml of serum-free medium for 30 min and was then overlaid with an additional 4.8 ml of Dulbecco's modified Eagle's medium for 5 h. Cells were then washed with phosphate-buffered saline and fed with serum-containing medium. After 48 h, cells were lysed in 1 ml of lysis buffer (20 mM Tris-Cl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 2.5 mM EDTA; 10 mM NaF; and 10 mM NaPP(i)) and subjected to immunoprecipitation.

Immunoprecipitation and Immunoblotting

The procedures for immunoblotting were essentially identical to those described previously (14) . In brief, an anti-murine c-met peptide antiserum directed against amino acids VAPYPSLLPSQDWIDGEGNT of the carboxyl terminus was raised in a rabbit. Transfected cells were lysed in the lysis buffer containing the protease inhibitors 1 mM phenylmethylsulfonyl fluoride (Sigma), 20 µg/ml aprotinin (Boehringer Mannheim), and 20 µg/ml leupeptin (Boehringer Mannheim), as well as 1 mM orthovanadate (Sigma). Lysates were immunoprecipitated with the above rabbit antiserum (1:25 dilution) plus 30 µl of protein G-coupled Sepharose (Pharmacia Biotech Inc.). The immunoprecipitates were washed three times with lysis buffer, solubilized with Laemmli buffer, boiled, and resolved by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA), and the blot was treated for 1 h with 3% nonfat dry milk in TTBS (Tris-buffered saline with 0.05% Tween 20), incubated with antiserum (1:200 dilution) in TTBS containing 0.5% bovine serum albumin (TTBS-BSA) for 1 h, and finally reacted with I-protein A (3 times 10^5 cpm/ml in TTBS-BSA) for 1 h. The blots were washed extensively between each treatment. After the final wash, the blot was air-dried and subjected to autoradiography using Kodak XAR film at -70 °C.


RESULTS

Transient Expression of HGF Receptor Isoforms in COS-7 Cells

To examine the function of the juxtamembrane domain in a regulatory signal transduction pathway, we constructed expression vectors with full-length cDNAs for both HGF receptor isoforms. Purified plasmid DNAs were used to transfect COS-7 cells to examine protein expression. Transiently expressed cells were harvested 48 h after transfection, and cell lysates were prepared as described under ``Materials and Methods.'' Equal amounts of cellular protein from the cell lysates were initially immunoprecipitated with the anti-c-met antibody. The immunoprecipitates were then Western blotted with the same antibody. As shown in Fig. 1A, both large (lane1) and small (lane2) forms of HGF receptors were expressed in the cells. The HGF receptor is known to be a heterodimeric protein consisting of 40-kDa (alpha chain) and 150-kDa (beta chain) subunits that are linked by disulfide bonds. Therefore, the upper bands of 190-kDa protein are the immature forms containing an additional 40 kDa, while the lower bands of 150 kDa represent the processed beta chain. The c-met antibody did not immunoprecipitate any endogenous HGF receptor in mock transfected COS-7 cells (lane3).


Figure 1: Detection of exogenous expressed HGF receptor isoforms by immunoblotting. Cell lysates were immunoprecipitated with c-met antibody followed by immunoblot analysis with the same antibody (A) or with anti-phosphotyrosine (alphaPY) antibody (B). Cells transfected with plasmid DNA of large and small HGF receptor isoforms are shown in lanes1 and 2, respectively, while mock transfection with control plasmid is shown in lane3 (A and B). Bands corresponding to HGF receptor isoforms are indicated by brackets in the center (HGFR). Protein size markers are indicated in kilodaltons (kd). IP, immunoprecipitate.



Signal Transduction Pathway Induced by Receptor Autophosphorylation

Since both receptor isoforms could be expressed independently in transfected cells, we examined whether the the presence or absence of the spliced juxtamembrane domain causes any specific alteration in total cellular tyrosine phosphorylation. Cell lysates of both isoform transfectants were prepared, and immunoprecipitations were performed using an anti-phosphotyrosine antibody. The phosphotyrosine profiles of total transfected as well as mock transfected cells were then analyzed. Western blotting with the anti-phosphotyrosine antibody was utilized to detect all the tyrosine-phosphorylated proteins in the cells. As shown in Fig. 2, A and B, cells transfected with cDNA of HGF receptors (lanes1 and 2) exhibited a series of phosphorylated proteins that are not seen in mock transfected cells (lane3). This result indicates that transfected HGF receptors in transiently expressed cells can exert signals involving cytoplasmic proteins. Importantly, a subtle but reproducible difference between the phosphotyrosine profiles was observed with the two isoforms. Phosphorylated proteins of 62 and 30 kDa were present in the total cellular protein of the large isoform transfectant (lane1 of Fig. 2, A and B) but not in that of the small form (lane2). Conversely, a phosphorylated protein of 35 kDa was observed in the total cellular protein of the small form but not the large form (Fig. 2B). Comparison of these phosphotyrosine profiles in transfected and mock transfected cells therefore suggests that transfection of exogenous HGF receptors induces phosphorylation of novel proteins compared with control cells and that the induction of phosphorylation of proteins by the two isoforms differs by at least three tyrosine-phosphorylated proteins.


Figure 2: Comparison of phosphotyrosine profile induced by receptor isoforms in transfected (lanes 1 and 2) and mock transfected (lane 3) COS-7 cells. The tyrosine-phosphorylated products that were differentially regulated by HGF receptor isoforms are indicated by arrows on the left. Protein size markers are shown on the right. IP, immunoprecipitate; alphaPY, anti-phosphotyrosine.



We next examined whether any of the tyrosine-phosphorylated proteins are receptor-associated proteins. The immunoprecipitates of c-met antibody were Western-blotted with anti-phosphotyrosine to detect tyrosine phosphorylation of c-met-precipitated proteins. As shown in Fig. 1B, both receptor isoforms underwent autophosphorylation, including the 190-kDa immature form; however, no additional tyrosine-phosphorylated protein was observed in this assay. This was confirmed by overexposing the x-ray film (data not shown). This result indicates that little or none of the tyrosine phosphorylated protein is associated with the HGF receptor.

Differential Binding of the p85 Subunit of PI(3) Kinase to HGF Receptor Isoforms

The presence of the juxtamembrane domain in the HGF receptor appears to be a positive factor for the induction of newly phosphorylated proteins. To investigate further the effect of the juxtamembrane domain on signaling proteins, we also compared quantities of certain signaling molecules in the c-metimmunoprecipitates. Surprisingly, we observed differential binding of the p85 subunit of the PI(3) kinase to the two receptor isoforms (Fig. 3A). The p85 subunit of the PI kinase showed much stronger association with the small isoform (lane2) than the large form (lane1). The binding of p85 to the HGF receptor, however, was dramatically inhibited by treatment with 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml) for 10 min (lane4), 1 h, 4 h, and 12 h (data not shown). To ensure the presence of HGF receptors in the cell lysates, the same blot was reprobed with anti-phosphotyrosine antibody without stripping the PI kinase signal (Fig. 3B). The residual p85 signal remaining from lane2 in Fig. 3A was seen to be with the small isoform (lane2 in Fig. 3B). This result suggests that the 47-amino acid sequence of the juxtamembrane domain has a negative effect on the binding of the p85 to the HGF receptor, since deletion of the region facilitates the binding of the p85 subunit to the receptor. Two other signaling proteins, GTPase-activating protein and phospholipase C, showed no differential affinity for the two isoforms (data not shown).


Figure 3: Association of p85 and HGF receptor isoforms in COS-7 transfected cells. Lysates of cells transfected with the HGF receptor isoforms were immunoprecipitated with c-met antibody followed by immunoblot analysis. The same blot was probed with anti-p85 antibody (UBI) (A) and then reprobed with anti-phosphotyrosine antibody (B). 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment at 10 min (lanes3 and 4) and controls (lanes1 and 2) are shown as indicated at the top. Bands corresponding to HGF receptor isoforms are indicated by brackets in the center (HGFR). Protein size markers are indicated in kilodaltons (kd). IP, immunoprecipitate; alphaPY, anti-phosphotyrosine.




DISCUSSION

We have previously identified a HGF receptor isoform that has a deletion of 47 amino acids in the juxtamembrane region. The removal of the 47-amino acid segment appears to occur by mRNA alternative splicing. The differential splicing occurs in a variety of mouse tissues, suggesting biological significance of the two isoforms. Removal of the alternatively spliced domain produces a tyrosine kinase receptor that lacks the protein kinase C target phosphorylation site and that would lack this mechanism for down-regulation of receptor kinase activity. To investigate additional roles of the juxtamembrane domain of the HGF receptor in cellular regulation, we used a transient expression system to express exogenous HGF receptor isoforms in COS-7 cells. The signaling proteins that were differentially regulated by receptor isoforms were analyzed by immunoblotting. We report here that at least three tyrosine-phosphorylated proteins are differentially induced by the receptor isoforms differing in the juxtamembrane region. In addition, studies using antisera to p85 of PI(3) kinase suggested that the 47-amino acid segment of the juxtamembrane domain is a negative controlling element for the binding of the p85 subunit of the PI(3) kinase, since deletion of this domain greatly enhanced the binding of PI(3) kinase to the receptor.

Our findings that the presence of the juxtamembrane domain has a significant effect on differential affinity of signaling proteins provides evidence that the juxtamembrane domain plays a potentially pivotal role in regulating signaling transduction pathways. Two newly phosphorylated proteins were specifically produced by the large isoform, i.e. in the presence of the juxtamembrane domain. The induction of this protein phosphorylation is presumably a further downstream signaling event, although the molecular mechanism is not clear. On the other hand, the juxtamembrane domain is also a negative controlling region for the binding of the PI(3) kinase to the HGF receptor. The autophosphorylated EGF receptor and HGF receptor bind weakly to PI(3) kinase in vivo(7, 19) . Compared with platelet-derived growth factor receptors, for instance, EGF receptors containing the juxtamembrane domain appear to show much weaker binding to PI(3) kinase even though the EGF receptor was overexpressed at 10 times or more higher levels than the platelet-derived growth factor receptor(19) . The evidence for complex formation of the PI(3) kinase with EGF receptor or HGFR involves detecting PI(3) kinase activity in vitro in anti-receptor immunoprecipitates or overexpressing either p85 subunit or the receptors. The binding of an intermediary protein(s) to the juxtamembrane domain may be involved in interrupting complex formation between PI(3) kinase and the receptor tyrosine kinase domain, since deletion of the region facilitates PI(3) kinase binding to the receptor. Alternatively, removal of the juxtamembrane domain may cause a conformational change in the receptor that facilitates its binding to PI(3) kinase.

Ras protein was recently shown to play an integral part in mediating the mobility signal of HGF/scatter factor(11) , suggesting involvement of Ras, PI(3) kinase, and activated HGF receptor in functional complex formation. Our finding that deletion of the juxtamembrane domain facilitates the binding of PI(3) kinase to the receptor clearly suggests that signals transmitted by each of the two HGF receptor isoforms may couple diverse signaling pathways and result in distinct cellular responses. Our results, therefore, provide the first direct evidence that a cytoplasmic juxtamembrane domain of the HGF receptor interacts with different signal transduction pathways. These findings may also partly explain the diverse biological functions of the HGF receptor.

Functional importance of the juxtamembrane region has also been reported for the EGF receptor(20) . Phosphorylation of a threonine residue by protein kinase C in the juxtamembrane region significantly decreases receptor kinase activity. A point mutation at the threonine residue in the sequence motif for protein kinase C phosphorylation prevents the receptor kinase from being down-regulated. The same regulatory mechanism involving protein kinase C was recently observed for the HGF receptor(15) . Within the juxtamembrane domain, however, there are also regions other than the protein kinase C target site that are important for functions of the receptor. A receptor mutant with a deletion (residues 660-667) six amino acids downstream from the protein kinase C regulatory site alters the mitogenic activity of the EGF receptor(21) . A point mutation within this region strongly affects the specificity of signal transduction, indicating that the juxtamembrane domain may impart specificity for certain cytoplasmic proteins(22) . In addition, the juxtamembrane region of the HGF receptor is also involved in transforming activity in focus formation and in nude mice(16) . These studies further strongly suggest that the juxtamembrane domain of kinase receptors has crucial and distinct biological and biochemical properties, underscoring the functional significance of its modification by alternative splicing.


FOOTNOTES

*
This work was supported in part by the Human Frontier Science Program. We thank Masatoshi Takeichi and Jean Paul Thiery for discussions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Cell Biology, School of Medicine, Georgetown University, Washington, D. C. 20007.

To whom correspondence should be addressed: Laboratory of Developmental Biology, NIDR, Bldg. 30, Rm. 423, NIH, Bethesda, MD 20892-4370. Tel.: 301-496-9124; Fax: 301-402-0897.

(^1)
The abbreviations used are: HGF, hepatocyte growth factor; PI(3) kinase, phosphatidylinositol-3-OH kinase; EGF, epidermal growth factor; HGFR, hepatocyte growth factor receptor.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.