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We describe a novel human adapter molecule containing a pleckstrin homolgy (PH) domain at the N terminus that is closely related to human Grb2-associatedbinder 1, Gab1, and Drosophila daughter of sevenless. We designate this protein as Gab2. Northern blot analysis indicates that Gab2 is widely expressed and has an overlapping but distinctive expression pattern as compared with Gab1, with high levels of Gab2 mRNA detected in the heart, brain, placenta, spleen, ovary, peripheral blood leukocytes, and spinal cord. Upon tyrosine phosphorylation, Gab2 physically interacts with Shp2 tyrosine phosphatase and Grb2 adapter protein. Strikingly, Gab2 has an inhibitory effect on the activation of Elk-1-dependent transcription triggered by a dominant active Ras mutant (RasV12) or under growth factor stimulation, whereas Gab1 acts to potentiate slightly the Elk-1 activity in the same system. In contrast to the reciprocal effects of Gab1 and Gab2 in mediating Elk-1 induction, these two molecules have a similar function in extracellular signal-regulated kinase activation induced by either oncogenic Ras or growth factor stimulation. Taken together, these results argue that Gab1 and Gab2, two closely related PH-containing adapter proteins, might have distinct roles in coupling cytoplasmic-nuclear signal transduction. This is the first evidence that an intracellular molecule with a PH domain operates as a negative effector in signal relay to the regulation of gene expression. We describe a novel human adapter molecule containing a pleckstrin homolgy (PH) domain at the N terminus that is closely related to human Grb2-associatedbinder 1, Gab1, and Drosophila daughter of sevenless. We designate this protein as Gab2. Northern blot analysis indicates that Gab2 is widely expressed and has an overlapping but distinctive expression pattern as compared with Gab1, with high levels of Gab2 mRNA detected in the heart, brain, placenta, spleen, ovary, peripheral blood leukocytes, and spinal cord. Upon tyrosine phosphorylation, Gab2 physically interacts with Shp2 tyrosine phosphatase and Grb2 adapter protein. Strikingly, Gab2 has an inhibitory effect on the activation of Elk-1-dependent transcription triggered by a dominant active Ras mutant (RasV12) or under growth factor stimulation, whereas Gab1 acts to potentiate slightly the Elk-1 activity in the same system. In contrast to the reciprocal effects of Gab1 and Gab2 in mediating Elk-1 induction, these two molecules have a similar function in extracellular signal-regulated kinase activation induced by either oncogenic Ras or growth factor stimulation. Taken together, these results argue that Gab1 and Gab2, two closely related PH-containing adapter proteins, might have distinct roles in coupling cytoplasmic-nuclear signal transduction. This is the first evidence that an intracellular molecule with a PH domain operates as a negative effector in signal relay to the regulation of gene expression. 3, Src homology 2, 3 phosphotyrosine binding pleckstrin homolgy glutathione S-transferase insulin receptor substrate extracellular signal-regulated kinase epidermal growth factor interleukin polymerase chain reaction hemagluttinin Molecular dissection of cytoplasmic signal transduction mechanisms has been greatly facilitated by the identification of a large group of signaling molecules with distinct structural motifs, such as Src homology 2 (SH2),1 SH3, phosphotyrosine binding (PTB), and pleckstrin homology (PH) domains (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2224) Google Scholar,2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (925) Google Scholar). These modules apparently mediate specific protein-protein or protein-lipid interactions and therefore orchestrate cellular responses to various extracellular stimuli. The PH domain was originally identified as a protein module of around 120 amino acids in a number of cytoplasmic signaling proteins that display homology to a region repeated in the protein pleckstrin (3Mayer B.J. Ren R. Clark K.L. Baltimore D. Cell. 1993; 73: 629-630Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 4Haslam R.J. Koide H.B. Hemmings B.A. Nature. 1993; 363: 309-310Crossref PubMed Scopus (387) Google Scholar, 5Tyers M. Rachubinski R.A. Stewart M.I. Varrichio A.M. Shorr R.G. Haslam R.J. Harley C.B. Nature. 1988; 333: 470-473Crossref PubMed Scopus (193) Google Scholar). Our current view on the function of PH domains stands on the point that they are involved in the recruitment of proteins to plasma membranes, possibly through binding to phosphatidylinositol polyphosphates (6Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). PH domains have been found in a variety of enzymes, such as Btk, Akt, βARK kinases, and phospholipase Cγ1, as well as in adapter molecules with no enzymatic activity that include Grb7 and insulin receptor substrate (IRS) 1–4 (7Myers Jr., M.G. White M.F. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 615-658Crossref PubMed Scopus (296) Google Scholar, 8Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 9Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Crossref PubMed Scopus (288) Google Scholar). In the latter subgroup, one attractive member identified recently is theGrb2-associated binder 1, Gab1 (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar). Structurally, Gab1 contains a PH domain at the N terminus and multiple tyrosyl residues at the C-terminal part, which presumably serve as docking sites for SH2-containing proteins in a tyrosine phosphorylation-dependent fashion. Overexpression of Gab1 stimulates cell growth and transformation (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar). Accumulating biochemical data suggest that Gab1 functions as a major adapter molecule downstream of receptors for nerve growth factor, hepatocyte growth factor, epidermal growth factor, insulin, and B cell antigen (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar, 11Holgado-Madruga M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar, 12Nguyen L. Holgado-Madruga M. Maroun C. Fixman E.D. Kamikura D. Fournier T. Charest A. Tremblay M.L. Wong A.J. Park M. J. Biol. Chem. 1997; 272: 20811-20819Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 13Fixman E.D. Holgado-Madruga M. Nguyen L. Kamikura D.M. Fournier T.M. Wong A.J. Park M. J. Biol. Chem. 1997; 272: 20167-20172Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 14Ingham R.J. Holgado-Madruga M. Siu C. Wong A.J. Gold M.R. J. Biol. Chem. 1998; 273: 30630-30637Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Genetic screening for suppressor mutations of a dominant active Sevenless protein in Drosophila led to the identification of the daughter of sevenless gene that also encodes for a PH-containing molecule, structurally related to mammalian Gab1 (15Raabe T. Riesgo-Escovar J. Liu X. Bausenwein B.S. Deak P. Maroy P. Hafen E. Cell. 1996; 85: 911-920Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Interestingly, daughter of sevenless seems to be a putative substrate for Corkscrew, a tyrosine phosphatase with two SH2 domains (16Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (330) Google Scholar), because a catalytically inactive mutant of Corkscrew trapped the daughter of sevenless protein in its highly tyrosine-phosphorylated form (17Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). It remains to be defined whether Gab1 is a physiological substrate of Shp2, the mammalian homologue of Corkscrew. However, it was demonstrated that Shp2 physically interacts with Gab1, and this interaction might play a critical role in transducing signals into the Ras pathway (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar, 14Ingham R.J. Holgado-Madruga M. Siu C. Wong A.J. Gold M.R. J. Biol. Chem. 1998; 273: 30630-30637Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 18Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell. Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (248) Google Scholar). In this paper, we report the identification of another Gab1-related molecule, designated as Gab2, that also contains a PH domain at the N terminus. Evidence is presented that although it acts to promote mitogenic stimulation of ERK kinase activity, Gab2 appears as a negative effector in the modulation of cytoplasmic signaling into the nucleus. These results allow us to make an intriguing proposal that a family of PH-containing adapter proteins functions in both negative and positive ways to control intracellular signaling cascades. A human cDNA clone was identified in the GenBankTM that shows high homology to the human Gab1 sequence (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar). This sequence was deposited by the Kazusa DNA Research Institute, Chiba, Japan and named as KIAA 0571 (clone: HH2388). A 6052-base pair fragment inserted at theSalI-NotI sites of pBluescript II SK+was kindly supplied to us by Dr. T. Nagase. In a comparison with the Gab1 sequence, this cDNA clone seemed to be incomplete at the 5′-end. Accordingly, a Human Testis Marathon-ReadyTMcDNA kit (CLONTECH) was used to amplify the 5′-end cDNA segment of Gab2. Primers and PCR parameters were designed according to the manufacturer's manual. Briefly, a touch-down program (5 cycles: 94 °C, 1 min; 72 °C, 2 min; 5 cycles: 94 °C, 1 min, 70 °C, 2 min; and 20–25 cycles: 94 °C, 1 min, 68 °C, 2 min) was used for all PCR reactions. The first round of PCR reaction was performed with primer AP1 from theCLONTECH kit and an antisense primer Z4 (5′-CAT GGA CTT GAC CAC TGA TCC CGT T-3′). The second round PCR used the primer AP1 and a nested antisense primer Z2 (5′-TGC TGA GCT CAG CTG GAG AAG AGC G-3′). The PCR product was then digested with SacI andNotI and cloned into Bluescript SK. All clones with inserts were sequenced either by Perkin Elmer/Applied Biosystems 377 XL DNA sequencer or by Thermo Sequenase Radiolabeled Terminator Cycle Sequencing System (Amersham Pharmacia Biotech). A composite Gab2 cDNA was generated by recombining a SacI-SacI fragment from KIAA0571 (480–1913) with a 5′-rapid amplification of cDNA ends product, clone C32 of 447 base pairs, which extends 12 base pairs at the 5′-end from the initiation codon ATG, as determined from comparison with Gab1. To engineer an HA-tagged Gab2 expression construct, the composite Gab2 cDNA was used as a template for PCR with a pair of primers: Z13 (5′-CGG GGT ACC ATG TAT GAT GTT CCT GAT TAT GCT AGC CTC ATG AGC GGC GGC GGC GAC GTG G-3′) and Z11 (5′-TAG GTA CTG TCT CTG AAT TCT G-3′). The PCR product was digested withKpnI and EcoRI and cloned into pcDNA3.1+ viaKpnI and BamHI sites, together with theEcoRI-BamHI fragment of KIAA0571 we previously made. The full-length construct was sequenced to confirm that no error had been introduced by PCR reaction. The mammalian expression construct for HA-tagged Gab1 was a gift from Drs. M. Hibi and T. Hirano at Osaka University (18Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell. Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (248) Google Scholar), and GST-tagged ERK1 was kindly provided by Dr. B. Mayer. α-HA (12CA5) antibody was from Roche Molecular Biochemicals, α-Shp2 and α-Grb2 antibodies and the mouse monoclonal anti-phosphotyrosine antibody (PY99) were from Santa Cruz Biotechnology, Inc. The expression of Gab2mRNA in various tissues was determined by using a Human Multiple Tissue Northern blot system purchased from previously R. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Northern was performed with from the manufacturer's DNA for were with using II kit from Human were in with in were with DNA by using the System A DNA fragment amino residues of Gab2 was cloned into protein was by To specific protein was into according to a at were in with and mouse were with and in for with for at were with and in X. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). were at for min, and were to with specific antibodies at for were with protein (Amersham Pharmacia at for were in and to proteins were to and the was in or were first with antibodies for in and with antibody for 1 were detected with the kit (Amersham Pharmacia Biotech). were and was by with (Amersham Pharmacia Biotech). The in ERK kinase was performed as previously J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The ERK1 was with The reaction was in of kinase 1 at for The were of was on The were with with and on a Human in were with which has the C-terminal domain of Elk-1 with the DNA binding domain of and which has the gene under the control of binding sites M. A. T. A. M. 1993; PubMed Scopus Google Scholar, R. Cell. 1995; 80: Full Text PDF PubMed Scopus Google Scholar). was also as an control to Elk-1 activation was by of cloned in the or epidermal growth factor stimulation B. D. A. S. A. M. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). the GenBankTM data we identified a human cDNA sequence that shows a high homology to Gab1. However, this cDNA fragment contains an for a of amino residues that seemed to a of the compared with Gab1. 5′-rapid amplification of cDNA we a number of cDNA that the of Gab2 to Upon recombining one 5′-rapid amplification of cDNA ends product with the KIAA0571 sequence, we a cDNA fragment that encodes a protein of amino on the amino sequence, this protein is of a PH domain at the N terminus and multiple tyrosine sites at the C-terminal part, enzymatic the of this molecule is closely related to human Gab1 daughter of sevenless (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar, T. Riesgo-Escovar J. Liu X. Bausenwein B.S. Deak P. Maroy P. Hafen E. Cell. 1996; 85: 911-920Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). We suggest Gab2 for the of this for results of Gab2 of sequence of the clone is with the region overlapping with KIAA0571 and the initiation codon ATG is in the Human Testis cDNA kit from we performed to for at the 5′-end of Gab2 cDNA and PCR at The sequence of the clone is with the region overlapping with KIAA0571 and the initiation codon ATG is in in a the Human Testis cDNA kit from we performed to for at the 5′-end of Gab2 cDNA and PCR at in 1 of the Gab2 PH domain with that of Gab1, daughter of and that Gab2 the homology to Gab1. The high sequence homology and the of Gab1, Gab2, and daughter of sevenless suggest that these proteins a of signal with a PH domain and multiple SH2 binding The family of proteins also a in to the PH domain and multiple tyrosine sites (7Myers Jr., M.G. White M.F. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 615-658Crossref PubMed Scopus (296) Google Scholar, 8Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 9Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Crossref PubMed Scopus (288) Google Scholar). It remains to be whether Gab1 Gab2 are mammalian daughter of sevenless. Gab1 and Gab2 are the sites the C-terminal of the molecule that have for binding SH2 domains M. T. T. S. R.J. B. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). are that are presumably involved in binding the SH2 domains of phosphatidylinositol the for phospholipase for and for Gab1, Gab2 two that are presumably in binding that is by Gab1, Gab2, and proteins is the of and are residues in Gab1 and in Gab2. This an that these adapter proteins might function to or signaling kinases, To the expression pattern of the human Gab2 we performed Northern blot analysis on various human in Gab2 is widely with high levels detected in the heart, brain, placenta, spleen, ovary, peripheral blood leukocytes, and spinal cord. with expression pattern previously (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar), is an for the expression of Gab1 and in such as heart, brain, spinal and expression of Gab1 and Gab2 was also in highly expressed in the and were levels of Gab2 mRNA the expression was in peripheral blood in which Gab2 was expressed at a high This expression pattern might the of Gab1 and Gab2 in overlapping distinct signaling in cell To the Gab2 function in cell we generated a specific antibody Gab2 by with a protein containing the C-terminal of Gab2. This antibody a protein of around Gab2 tyrosine-phosphorylated and with Shp2 tyrosine phosphatase and Grb2 adapter protein in with as detected by We also the to of and expressed in human of for induced a in the tyrosine of Gab1 and Gab2 demonstrated in 1 are two in Gab2, which might be in protein To this we performed an in binding on the interaction of expressed in with proteins that These were from a number of signaling and phospholipase as well as full-length in the domain of Grb2 has the to Gab2, whereas were levels of binding for Gab2 with the Grb2 SH3, phospholipase SH3, and This that Grb2 might be a of Gab2 in a that It is to that the binding of Grb2 to Gab2 is apparently from the which is through the domain of Grb2 M. R. J. T. D. Nature. 1993; 363: PubMed Scopus Google Scholar, S. Schlessinger J. D. Nature. 1993; 363: PubMed Scopus Google Scholar, L. J. Cell. 1993; 73: Full Text PDF PubMed Scopus Google Scholar). and biochemical on Gab2 suggest that this novel PH protein might in the Ras signaling with similar function to Gab1. To this we compared the effect of Gab1 and Gab2 on the activation of by the activity under the control of a member of the Human were with Gab1 or Gab2, and Gab1 and Gab2 had effects in the modulation of Elk-1 Gab1 had a effect on Elk-1-dependent transcription in to the dominant active Gab2 a of Elk-1 activity under the same To its inhibitory we the activity under growth factor stimulation. were with and together with Gab1 or Gab2. were for 12 and with human for Gab2, in contrast to Gab1, had a activity on Elk-1 function under stimulation 5 To the expression levels of and in cell were with and similar protein of Gab1 and Gab2 were detected 5 Taken together, these results that Gab2 acts to Ras signaling into the in the modulation of Elk-1 transcription The results a that Gab1 and Gab2, two structurally related might have effects in mediating the Ras signal that is presumably through ERK kinase into the nucleus. To the we the of Gab1 and Gab2 on activation of the ERK kinase by were with or and was with from cell and ERK activity was in an in kinase using protein as a in expression of in induced a in the ERK1 This effect was slightly by of either Gab1 or Gab2, that these two molecules have similar roles in mediating the Ras to ERK To this we compared the effect of Gab1 and Gab2 on ERK in expression of Gab1 or Gab2 had a similar and effect on ERK1 These results suggest that Gab1 and Gab2 in the same in the signaling by growth factor or oncogenic However, Gab2 apparently functions as a negative effector to the signal of the pathway in the control of gene expression. We have a novel adapter Gab2, that contains a PH domain at the N terminus and the same as Gab1. Gab2 is widely expressed in human We also a interaction Gab2 and Shp2, The domain of Grb2 has a high binding Gab2. Gab2 has a positive effect similar to Gab1 in mediating the of ERK kinase activity by growth factor or oncogenic However, Gab2 appears to function in a in Ras signaling by Elk-1 activation from ERK kinase activity, in contrast to the positive role of Gab1 in the same the physiological function of Gab1 has been it apparently acts to promote cell growth and Overexpression of Gab1 to cell and cell growth in (10Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar). of Gab1 and its with Grb2 might also play an role in cellular transformation by an of the domain of hepatocyte growth factor receptor tyrosine kinase with by the gene E.D. Holgado-Madruga M. Nguyen L. Kamikura D.M. Fournier T.M. Wong A.J. Park M. J. Biol. Chem. 1997; 272: 20167-20172Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). A mutant protein that to to Grb2 has it is Gab1 functions in cytoplasmic the of this protein that Gab1 is tyrosine at sites and therefore is in the of a that of proteins, phospholipase Shp2, and phosphatidylinositol In this Gab1 and Gab2 might in in a similar as the family of insulin receptor by coupling to multiple signaling However, the is the of a domain the PH domain in the proteins, which is from Gab1 and Gab2. a might with growth factor receptors SH2-containing such as Gab1 and Gab2 structural with daughter of sevenless protein (15Raabe T. Riesgo-Escovar J. Liu X. Bausenwein B.S. Deak P. Maroy P. Hafen E. Cell. 1996; 85: 911-920Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). is daughter of sevenless physically with Corkscrew, but daughter of sevenless is the major trapped by a catalytically inactive mutant of Corkscrew that was in (17Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Gab1 and Gab2 have been detected in with Shp2, the mammalian homologue of Drosophila Corkscrew. the critical is to whether they are the physiological of Shp2 and this to the Ras signaling It is that these two molecules serve as of the expressed Shp2 phosphatase in cell these proteins also be of another which is expressed in T. Full Text PDF PubMed Scopus Google Scholar, Biol. 1997; PubMed Scopus Google Scholar). The in this report is the reciprocal effects of Gab1 and Gab2 in mediating Elk-1 activation in Gab2 to the ERK kinase activation by either receptor tyrosine kinase or dominant active but it acts to signals from ERK to a member of this was under Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google the of mouse with expression of in also to a of Elk-1 activity, whereas ERK1 kinase activation was This inhibitory effect of mouse Gab2 on Elk-1 activity was by the of a C-terminal that its binding to A similar effect on ERK kinase to Elk-1 stimulation was recently for the kinase suppressor of Ras T. Stewart S. M. K.L. J. 1998; PubMed Scopus Google Scholar). the kinase suppressor of Ras and the Ras of Elk-1-dependent the Gab2 and kinase suppressor of Ras might of a novel and pathway that the signal relay from the kinase to the activation of the for its on Elk-1 activation is to be the Gab2 protein acts apparently by with enzymes, such as and that SH2 and of the function of Gab2 into the by which the is We are to Dr. and at the Kazusa DNA Research for the gift of cDNA clone We also Drs. S. M. B. D. M. L. and R. for or critical of the
Zhao et al. (Thu,) studied this question.