Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria toxin receptor-associated protein (DRAP27)/CD9 form a complex with integrin alpha 3 beta 1 at cell-cell contact sites

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF receptor to exert mitogenic activity. The membrane-anchored form of HB- EGF, proHB-EGF, is biologically active, providing mitogenic stimulation to neighboring cells in a juxtacrine mode. ProHB-EGF forms a complex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a tetra membrane-spanning protein that upregulates the juxtacrine mitogenic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/CD9 resulted in preferential coprecipitation of integrin alpha 3 beta 1 from Vero cell, A431 cell and MG63 cell lysates. Anti-integrin alpha 3 or anti-integrin beta 1 coprecipitated DRAP27/CD9 from the same cell lysates. Chemical cross-linking confirmed the physical association of DRAP27/CD9 and integrin alpha 3 beta 1. Using Vero-H cells, which overexpress HB-EGF, we also demonstrated the association of proHB-EGF with DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contact sites was observed by double-immunofluorescence staining. At cell-cell contact sites, DRAP27/CD9 was highly coincident with alpha- catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alpha 3 beta 1 are colocalized with adherence junction- locating proteins. These results indicate that direct interaction of growth factors and cell adhesion molecules may control cell proliferation during the cell-cell adhesion process.     

Possible role of coexpression of CD9 with membrane-anchored heparin-binding EGF-like growth factor and amphiregulin in cultured human keratinocyte growth

CD9 is a protein with 4 transmembrane domains, and functions as a cell surface antigen. We have previously reported that CD9 functions as an up-regulator of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) activity, which is a potent mitogen as well as a soluble HB-EGF. Anti-CD9 antibodies can neutralize the juxtacrine activity of proHB-EGF when both CD9 and proHB-EGF are coexpressed. We demonstrated here: (1) the CD9 gene was transcribed and translated in the cultured human keratinocytes; (2) anti-CD9 antibody inhibited the approximately 50% growth of human keratinocytes in culture; (3) CD9 was coprecipitated with proHB-EGF and membrane-anchored amphiregulin (proAR), and (4) the transient coexpression of CD9 with proHB-EGF or proAR in mouse L cells up-regulated their juxtacrine growth factor activities. These results suggest that CD9 would make a heterodimer and/or trimer complex with proHB-EGF and proAR, and might cooperate with proHB-EGF and proAR for human keratinocyte growth in a juxtacrine manner. J. Cell. Physiol. 171:291–298, 1997. © 1997 Wiley-Liss, Inc.     

Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.

Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.     

Importance of the major extracellular domain of CD9 and the epidermal growth factor (EGF)-like domain of heparin-binding EGF-like growth factor for up-regulation of binding and activity

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors. The membrane-anchored form of HB-EGF (proHB-EGF) is mitogenically active to neighboring cells as well as being a precursor of the soluble form. In addition to its mitogenic activity, proHB-EGF has the property of binding to diphtheria toxin (DT), serving as the specific receptor for DT. Tetramembrane-spanning protein CD9, a member of the TM4 superfamily, is physically associated with proHB-EGF at the cell surface and up-regulates both mitogenic and DT binding activities of proHB-EGF. To understand this up-regulation mechanism, we studied essential regions of both CD9 and proHB-EGF for up-regulation. Immunoprecipitation experiments revealed that not only CD9 but also other TM4 proteins including CD63, CD81, and CD82 associate with proHB-EGF on the cell surface. However, these TM4 proteins did not up-regulate DT binding activity of proHB-EGF. Transfection of a series of chimeric constructs comprising CD9 and CD81 showed that the major extracellular domain of CD9 is essential for up-regulation. Assays of DT binding activity and juxtacrine mitogenic activity of the deletion mutants of proHB-EGF and chimeric molecules, derived from proHB-EGF and TGF-alpha, showed that the essential domain of proHB-EGF for up-regulation is the EGF-like domain. These results indicate that the interaction of the extracellular domains of both molecules is important for up-regulation.     

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin- sensitive cells

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.     

Domain analysis of the tetraspanins: studies of CD9/CD63 chimeric molecules on subcellular localization and upregulation activity for diphtheria toxin binding

CD9 and CD63 belong to a tetramembrane-spanning glycoprotein family called tetraspanin, and are involved in a wide variety of cellular processes, but the structure-function relationship of this family of proteins has yet to be clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activity in spite of the fact that this protein also associates with DTR/proHB-EGF on the cell surface. CD9 molecules localize on the cell surface, while those of CD63 localize predominantly at lysosomes and intracellular compartments. We made CD9/CD63 chimeric molecules and then studied their intracellular localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effect on the expression of the chimeric proteins at the cell surface, while mutants lacking the lysosome sorting motif delivered more efficiently on the cell surface, indicating that the lysosome sorting motif contributes to the inhibitory effect of the C-terminal region. However, the N-terminal half of this family of proteins containing the 1st to 3rd transmembrane domains also seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essential, while the remaining regions influenced the upregulation activity by changing the efficiency of cell surface expression. From these results we discussed the structure-function relationship of this family of proteins.     

Heparin-binding EGF-like growth factor: a juxtacrine growth factor

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family growth factors, is synthesized as a membrane-anchored form (proHB-EGF). Proteolytic cleavage of proHB-EGF at the extracellular domain yields the soluble form of HB-EGF (sHB-EGF). ProHB-EGF is not only the precursor molecule for sHB-EGF but also a biologically active molecule itself. Recent studies indicate that proHB-EGF has unique properties distinct from the soluble form. ProHB-EGF forms a complex with membrane proteins including a tetramembrane spanning protein: CD9, an adhesion molecule integrin: 3β1, and heparan sulfate proteoglycans. The complex is localized at the cell–cell contact site, suggesting that proHB-EGF may function in cell-to-cell signaling by a juxtacrine mechanism. In an in vitro model system, proHB-EGF showed growth inhibitory activity, while sHB-EGF was growth stimulatory. Ectodomain shedding, conversion of the membrane-anchored form into the soluble form, is regulated by multiple signaling pathways. All these characteristics imply that proHB-EGF and sHB-EGF are used in different ways. In vivo functions of sHB-EGF and proHB-EGF have been largely undefined, but recent studies implicate them in a variety of physiological processes including blastocyst implantation and wound healing.     

Roles of Dppa2 in the regulation of the present status and future of pluripotent stem cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored protein, known as proHB-EGF. ProHB-EGF is cleaved by metalloproteases through a process referred to as 'ectodomain shedding', resulting in the formation of soluble HB-EGF. Both proHB-EGF and soluble HB-EGF are biologically active; the former acts on neighbouring cells through juxtacrine signalling, whereas the latter can move to distant locations. Elevated HB-EGF expression has been observed in ovarian and some other cancers. CRM197, a diphtheria toxin (DT) mutant, binds directly to the epidermal growth factor (EGF)-like domain and represses the mitogenic activity of HB-EGF. Recently, monoclonal antibodies (mAbs) specific for human HB-EGF were generated by immunizing HB-EGF-deficient mice with human HB-EGF (Hamaoka et al. (2010) J. Biochem. 148, 55-69). Most of the mAbs can bind to the EGF-like domain of HB-EGF, but fail to inhibit the mitogenic activity of soluble HB-EGF. However, some mAbs prevented the ectodomain shedding of proHB-EGF and inhibited the proliferation of EGF receptor-expressing cells stimulated by proHB-EGF-expressing cells. Hamaoka et al. showed that CRM197 prevents the ectodomain shedding of proHB-EGF. Thus, these mAbs function as specific inhibitors for the ectodomain shedding of HB-EGF and may be useful for treating cancers exhibiting elevated levels of HB-EGF.     

Heparin-binding EGF-like growth factor in the human prostate: Synthesis predominantly by interstitial and vascular smooth muscle cells and action as a carcinoma cell mitogen

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an activating ligand for the EGF receptor (HER1/ErbB1) and the high-affinity receptor for diphtheria toxin (DT) in its transmembrane form (proHB-EGF). HB-EGF was immunolocalized within human benign and malignant prostatic tissues, using monospecific antibodies directed against the mature protein and against the cytoplasmic domain of proHB-EGF. Prostate carcinoma cells, normal glandular epithelial cells, undifferentiated fibroblasts, and inflammatory cells were not decorated by the anti-HB-EGF antibodies; however, interstitial and vascular smooth muscle cells were highly reactive, indicating that the smooth muscle compartments are the major sites of synthesis and localization of HB-EGF within the prostate. In marked contrast to prostatic epithelium, proHB-EGF was immunolocalized to seminal vesicle epithelium, indicating differential regulation of HB-EGF synthesis within various epithelia of the reproductive tract. HB-EGF was not overexpressed in this series of cancer tissues, in comparison to the benign tissues. In experiments with LNCaP human prostate carcinoma cells, HB-EGF was similar in potency to epidermal growth factor (EGF) in stimulating cell growth. Exogenous HB-EGF and EGF each activated HER1 and HER3 receptor tyrosine kinases and induced tyrosine phosphorylation of cellular proteins to a similar extent. LNCaP cells expressed detectable but low levels of HB-EGF mRNA; however, proHB-EGF was detected at the cell surface indirectly by demonstration of specific sensitivity to DT. HB-EGF is the first HER1 ligand to be identified predominantly as a smooth muscle cell product in the human prostate. Further, the observation that HB-EGF is similar to EGF in mitogenic potency for human prostate carcinoma cells suggests that it may be one of the hypothesized stromal mediators of prostate cancer growth. J. Cell. Biochem. 68:328-338, 1998. © 1998 Wiley-Liss, Inc.     

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.     

CD9 amino acids critical for upregulation of diphtheria toxin binding.

CD9 associates with a diphtheria toxin receptor (DTR) that is identical to the membrane-anchored form of heparin-binding EGF-like growth factor. We determined the region of CD9 important for upregulation activity. Human and monkey CD9 upregulates DT binding activity of DTR, while mouse CD9 has no upregulation activity. Transfection of chimeric constructs comprising monkey and mouse CD9s showed that the human sequence between Ala156 and Asp183 is essential for the upregulation activity. Studies of mutants, replacing a single amino acid within the region between Ala156 and Asp183 of monkey CD9 with the corresponding amino acid residue in mouse CD9, revealed that substitution of Gly158 is critical for the reduction of the upregulation activity and secondly for the substitution of Val159 and Thr175. These three amino acid residues were deduced to be located on the head domain of the second extracellular loop, suggesting that interactions of CD9 with DTR or DT at the domain containing these three amino acids were important for the upregulation of DT binding.     

Expression of a transmembrane phosphotyrosine phosphatase inhibits cellular response to platelet-derived growth factor and insulin-like growth factor-1.

Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.     

Human platelet lysate contains growth factor activities for established cell lines derived from various tissues of several species

Summary Factors have been studied from human platelets that promote the growth of a hormone-responsive rat mammary adenocarcinoma cell line MTW9/PL, the BALB/c 3T3 mouse embryo fibroblasts, and numerous other established cell lines. A wide variety of the commonly employed cell lines, including lines of human, mouse, monkey, chicken, rat, Chinese hamster, and Syrian hamster origin, were tested for their growth response to a standard concentration of 200 μg/ml human platelet lysate, and the lysate was found to contain mitogenic activity for 24 of the 29 different lines assayed. A comparison was made between the platelet growth activity for the MTW9/PL cells and the well characterized platelet mitogen for the BALB/c 3T3 cells, platelet-derived growth factor (PDGF). When the platelet lysate was subjected to digestion by highly purified trypsin, the mitogenic activity for the MTW9/PL cells was not affected whereas that for the BALB/c 3T3 cells was essentially destroyed. Crude PDGF was prepared by heating the human platelet lysates at 100°C for 2 min followed by clarification, dialysis, lyophilization, and reconstitution. This PDGF material had no apparent growth activity for MTW9/PL cells, although chromatography of this material on Biogel P-100 revealed a high molecular weight (approximately 40,000 daltons) activity for the BALB/c 3T3 cells (presumably PDGF) and two growth activities for the MTW9/PL cells, one high molecular weight activity and a second activity of molecular weight less than 10,000. These studies demonstrated a form of epithelial tumor cell growth activity separable from the 3T3 type PDGF in crude heated extracts. This work was supported by American Cancer Society Grant BC-255B and NIH Grant CA 26617.     

Identification of the Cancer Cell Proliferation and Survival Functions of proHB-EGF by Using an Anti-HB-EGF Antibody

Purpose

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The membrane-bound proHB-EGF is known to be a precursor of the soluble form of HB-EGF (sHB-EGF), which promotes cell proliferation and survival. While the functions of sHB-EGF have been extensively studied, it is not yet fully understood if proHB-EGF is also involved in cellular signaling events. In this study, we utilized the anti-HB-EGF monoclonal antibodies Y-142 and Y-073, which have differential specificities toward proHB-EGF, in order to elucidate proHB-EGF functions in cancer cells.

Experimental Design

The biological activities of proHB-EGF were assessed in cell proliferation, caspase activation, and juxtacrine activity assays by using a 3D spheroid culture of NUGC-3 cells.

Results

Y-142 and Y-073 exhibited similar binding and neutralizing activities for sHB-EGF. However, only Y-142 bound to proHB-EGF. We could detect the function of endogenously expressed proHB-EGF in a 3D spheroid culture. Blocking proHB-EGF with Y-142 reduced spheroid formation, suppressed cell proliferation, and increased caspase activation in the 3D spheroid culture of NUGC-3 cells.

Conclusions

Our results show that proHB-EGF acts as a cell proliferation and cell survival factor in cancer cells. The results suggest that proHB-EGF may play an important role in tumor progression.     

The tetraspanin CD9 associates with transmembrane TGF-alpha and regulates TGF-alpha-induced EGF receptor activation and cell proliferation

Transforming growth factor-alpha (TGF-alpha) is a member of the EGF growth factor family. Both transmembrane TGF-alpha and the proteolytically released soluble TGF-alpha can bind to the EGF/TGF-alpha tyrosine kinase receptor (EGFR) and activate the EGFR-induced signaling pathways. We now demonstrate that transmembrane TGF-alpha physically interacts with CD9, a protein with four membrane spanning domains that is frequently coexpressed with TGF-alpha in carcinomas. This interaction was mediated through the extracellular domain of transmembrane TGF-alpha. CD9 expression strongly decreased the growth factor- and PMA- induced proteolytic conversions of transmembrane to soluble TGF-alpha and strongly enhanced the TGF- alpha-induced EGFR activation, presumably in conjunction with increased expression of transmembrane TGF-alpha. In juxtacrine assays, the CD9-induced EGFR hyperactivation by transmembrane TGF-alpha resulted in increased proliferation. In contrast, CD9 coexpression with transmembrane TGF-alpha decreased the autocrine growth stimulatory effect of TGF-alpha in epithelial cells. This decrease was associated with increased expression of the cdk inhibitor, p21(CIP1). These data reveal that the association of CD9 with transmembrane TGF-alpha regulates ligand-induced activation of the EGFR, and results in altered cell proliferation.     

Toxin binding site of the diphtheria toxin receptor: loss and gain of diphtheria toxin binding of monkey and mouse heparin-binding, epidermal growth factor-like growth factor precursors by reciprocal site-directed mutagenesis

The transmembrane precursor of the monkey (Mk) heparin-binding, epidermal growth factor-like growth factor (proHB-EGF) functions as a diphtheria toxin (DT) receptor, whereas the mouse (Ms) precursor does not. Previously, using chimeric Ms/Mk precursors, we have shown that DT resistance of cells bearing Ms proHB-EGF may be accounted for by several amino acid substitutions between residues 122 and 148 within the EGF-like domain and that Glu-141 is an important amino acid residue for DT binding. In this study, reciprocal site-directed mutagenesis was performed on the major non-conserved residues in the region of 122–148, alone or in combination, between Mk and Ms precursors to identify more precisely which amino acid residues are important for DT binding. Two approaches were used. The first, more traditional approach was to destroy DT sensitivity and binding of Mk proHB-EGF by substitution(s) with the corresponding Ms residue(s). From the single mutations, the greatest loss of DT sensitivity was observed with Mk/Glu-141His (approximately 4000-fold) and the next greatest with Mk/Ile-133Lys (approximately fourfold). The double mutations Mk/Leu-127Phe/Glu-141His, Mk/Ile-133Lys/Glu-141His and Mk/His-135Leu/Glu-141His resulted in complete toxin resistance (> 100 000-fold). The second approach, both novel and complementary, was to gain DT binding and sensitivity of Ms proHB-EGF by substitution(s) with the corresponding Mk residue(s). Surprisingly, the single mutation Ms/His-141Glu resulted in the gain of moderate DT sensitivity (> 260-fold). The double mutation Ms/Lys-133Ile/His-141Glu and the triple mutation Ms/Lys-133Ile/Leu-135His/His-141Glu resulted in a progressive gain in toxin sensitivity (> 4700-fold and > 16 000-fold respectively) and affinity. This triple mutant cell line is essentially as sensitive (IC₅₀ = 3.1 ng ml^?1) as the highly toxin-sensitive monkey Vero cell line (IC₅₀ = 4 ng ml^?1), indicating that these three Mk residues enable the Ms proHB-EGF to act as a fully functional DT receptor. Taken together, these results indicate that Glu-141 plays the most critical role in DT binding and sensitivity and that two additional amino acid residues, Ile-133 and His-135, also play significant roles.