Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform

Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine residues present in C1, and we determined that the loss of Tyr-770 alone enhanced FGFR2 IIIb C1 transforming activity. Because Tyr-770 may compose a putative YXXL sorting motif, we hypothesized that loss of Tyr-770 in the 770YXXL motif may cause disruption of FGFR2 IIIb C1 internalization and enhance transforming activity. Surprisingly, we found that mutation of Leu-773 but not Tyr-770 impaired receptor internalization and increased receptor stability and activation. Interestingly, concurrent mutations of Tyr-770 and Leu-773 caused 2-fold higher transforming activity than caused by the Y770F or L773A single mutations, suggesting loss of Tyr and Leu residues of the 770YXXL773 motif enhances FGFR2 IIIb transforming activity by distinct mechanisms. We also determined that loss of Tyr-770 caused persistent activation of FRS2 by enhancing FRS2 binding to FGFR2 IIIb. Furthermore, we found that FRS2 binding to FGFR2 IIIb is required for increased FRS2 tyrosine phosphorylation and enhanced transforming activity by Y770F mutation. Our data support a dual mechanism where deletion of the 770YXXL773 motif promotes FGFR2 IIIb C3 transforming activity by causing aberrant receptor recycling and stability and persistent FRS2-dependent signaling. Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine residues present in C1, and we determined that the loss of Tyr-770 alone enhanced FGFR2 IIIb C1 transforming activity. Because Tyr-770 may compose a putative YXXL sorting motif, we hypothesized that loss of Tyr-770 in the 770YXXL motif may cause disruption of FGFR2 IIIb C1 internalization and enhance transforming activity. Surprisingly, we found that mutation of Leu-773 but not Tyr-770 impaired receptor internalization and increased receptor stability and activation. Interestingly, concurrent mutations of Tyr-770 and Leu-773 caused 2-fold higher transforming activity than caused by the Y770F or L773A single mutations, suggesting loss of Tyr and Leu residues of the 770YXXL773 motif enhances FGFR2 IIIb transforming activity by distinct mechanisms. We also determined that loss of Tyr-770 caused persistent activation of FRS2 by enhancing FRS2 binding to FGFR2 IIIb. Furthermore, we found that FRS2 binding to FGFR2 IIIb is required for increased FRS2 tyrosine phosphorylation and enhanced transforming activity by Y770F mutation. Our data support a dual mechanism where deletion of the 770YXXL773 motif promotes FGFR2 IIIb C3 transforming activity by causing aberrant receptor recycling and stability and persistent FRS2-dependent signaling. Fibroblast growth factors (FGFs) 2The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; RTK, receptor tyrosine kinase; KGF, keratinocyte growth factor; PLCγ, phospholipase C gamma; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; FRS2, FGF receptor substrate 2; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PI, phosphatidylinositol; WT, wild type; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase. compose a large family of structurally related growth factors (22 human members) that mediate cell proliferation, differentiation, migration, and angiogenesis (1Dailey L. Ambrosetti D. Mansukhani A. Basilico C. Cytokine Growth Factor Rev. 2005; 16: 233-247Crossref PubMed Scopus (530) Google Scholar, 2Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1495) Google Scholar, 3Grose R. Dickson C. Cytokine Growth Factor Rev. 2005; 16: 179-186Crossref PubMed Scopus (334) Google Scholar, 4Mohammadi M. Olsen S.K. Ibrahimi O.A. Cytokine Growth Factor Rev. 2005; 16: 107-137Crossref PubMed Scopus (557) Google Scholar). The activities of FGFs are mediated by their binding to a family of four receptor tyrosine kinases (RTKs), designated FGFR1–4 (5Itoh N. Ornitz D.M. Trends Genet. 2004; 20: 563-569Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar). FGFRs are composed of an extracellular domain that consists of two or three Ig-like domains, a single transmembrane domain, and an intracellular catalytic tyrosine kinase domain and flanking regulatory sequences (Fig. 1A). An important feature and mode of regulation of FGFR2 function is that structural variants of FGFR2 are generated by numerous alternative gene splicing events that generate transcripts that encode proteins altered in both the extracellular and intracellular regions of the FGFR2 (5Itoh N. Ornitz D.M. Trends Genet. 2004; 20: 563-569Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar). To date, more than 20 alternative splicing variants of FGFR2 have been identified. The first major splicing event occurs in the second half of the third Ig-like domain (designated Ig-III domain). Tissue-specific inclusion of IIIb or that for the second half of the Ig-III domain the IIIb or isoforms A. A. D. PubMed Scopus Google Scholar). alternative splicing the binding of FGFR2 IIIb and but not FGFR2 but not and J. Full Text PDF PubMed Google Scholar, M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, A. A. D. J. PubMed Scopus Google Scholar). Interestingly, FGFR2 IIIb expression is to A. A. D. PubMed Scopus Google expression of the for FGFR2 IIIb and is to D. PubMed Scopus Google Scholar, A. A. M. N. PubMed Google Scholar, A. PubMed Scopus Google Scholar, M. A. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, R. Dickson C. PubMed Scopus Google in the of a in that is to for FGFR2 IIIb activity in The second major splicing occurs in sequences that encode the carboxyl of splice variants of FGFR2 IIIb that in their carboxyl-terminal sequences have been (designated C1, C2, and M. M. Google Scholar). The carboxyl is than the carboxyl and the carboxyl is than carboxyl (Fig. 1A). in of tyrosine residues that may of receptor and for cytoplasmic found that expression of the C3 increased in cell M. M. Google Scholar). We also enhanced expression of C2 and C3 isoforms in a of human cell with human PubMed Scopus Google suggesting that aberrant expression of the C2 or C3 splicing variants may to Furthermore, the C3 variant that lacks carboxyl-terminal sequences to more transforming than the IIIb C1 variant in and human M. M. Google Scholar, 2004; Google Scholar). the C2 variant is more transforming than the C1 variant not been Furthermore, the for the enhanced transforming activity of the C3 variant to FGFRs are by causing of their tyrosine kinase tyrosine and of proteins to tyrosine residues in their cytoplasmic carboxyl The two of FGFRs are phospholipase and FGF receptor substrate 2 (1Dailey L. Ambrosetti D. Mansukhani A. Basilico C. Cytokine Growth Factor Rev. 2005; 16: 233-247Crossref PubMed Scopus (530) Google Scholar, 2Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1495) Google Scholar, 3Grose R. Dickson C. Cytokine Growth Factor Rev. 2005; 16: 179-186Crossref PubMed Scopus (334) Google Scholar, 4Mohammadi M. Olsen S.K. Ibrahimi O.A. Cytokine Growth Factor Rev. 2005; 16: 107-137Crossref PubMed Scopus (557) Google Scholar). in the carboxyl is the major and a binding for the 2 domain of PLCγ, in tyrosine phosphorylation and activation of M. N. M. Schlessinger J. PubMed Scopus Google Scholar, M. D. R. M. M. Schlessinger J. PubMed Google to of phosphatidylinositol and the of and Rev. PubMed Scopus Google Scholar). The in is in four family and to Tyr-770 in FGFR2 IIIb (Fig. that a Y770F of FGFR2 IIIb C1 and M. 2005; PubMed Scopus Google Scholar). to and altered binding and activation to FGFR2 transforming activity PLCγ, FRS2 with is and of and receptor phosphorylation N. Schlessinger J. Lax I. 20: PubMed Scopus Google Scholar). FRS2 is a protein that to a the domain of activation of FGFRs to phosphorylation of tyrosine residues in FRS2 J. D. Lax I. Schlessinger J. Full Text Full Text PDF PubMed Scopus Google Scholar). FRS2 is the protein that to FRS2 and a with the activation of the and the J. D. Lax I. Schlessinger J. Full Text Full Text PDF PubMed Scopus Google Scholar). the protein is by FGFR2 activation. The phosphatidylinositol in activation of the kinase N. Schlessinger J. Lax I. A. PubMed Scopus Google Scholar). mechanism of regulation of receptor internalization and J. 2004; PubMed Scopus Google Scholar, I. PubMed Scopus Google Scholar). disruption of receptor internalization to aberrant receptor activation and to the of human Interestingly, FGFR2 IIIb C1 a putative sorting motif is and is a Rev. PubMed Scopus Google in carboxyl-terminal sequences in the C3 suggesting that the loss of the motif receptor internalization and to the enhanced transforming activity of the FGFR2 IIIb C3 To date, a of the transforming of FGFR2 IIIb C1, C2, and C3 the cell not been we the transforming of FGFR2 IIIb C1, C2, and C3 splicing variants and found that the C3 variant is more transforming than the C2 and the C2 variant is enhanced in transforming activity with the transforming C1 Our support a where the transforming activity of the FGFR2 IIIb C3 splice variant is in by a mechanism loss of the motif, in impaired receptor internalization and enhanced FRS2-dependent signaling. FGFR2 IIIb C1, C2, and C3 sequences generated by a cell and the expression and The FGFR2 IIIb C2 of FGFR2 IIIb C1 residues by the FGFR2 IIIb C1 and and sequences mutations by the and by to the mutations to encode the are and and and in with and with wild FGFR2 IIIb isoforms and of FGFR2 IIIb of FGFR2 IIIb C1 proteins determined by with in growth with and and in growth for and with To growth the or FGFR2 IIIb proteins in in growth with 2 and the of fibroblast and a with the or or Y770F FGFR2 IIIb C1, in or growth 2 for and with that wild or FGFR2 IIIb proteins for 20 and with serum or for to in phosphate-buffered and with FGFR2 to FGFR2 IIIb by of FGFR2 IIIb M. L. R. J. PubMed Scopus Google with the that wild or FGFR2 IIIb proteins for 20 The three with and with for three with and with for the to and three with To the three for with BSA, and with and for with with to five and with by with FGFR2 by of that wild or FGFR2 IIIb proteins with for the to receptor with FGFR2 to the of FGFR2 by FGFR2 tyrosine and for FGFR2 by with an by with an To and for by with by with to activity we have Google Scholar). wild FGFR2 IIIb C1 or with in with or for 20 in with of Google Scholar). To FRS2 and for FRS2 by with by with To and for by with by with To MEK, ERK, or and for MEK, ERK, or by with to or and determined by and To FRS2 binding to and with by with The and with the to the protein of of FGFR2 IIIb in and the of carboxyl-terminal sequences in FGFR2 IIIb we the transforming of FGFR2 IIIb C1, C2, and C3 variants by two of growth loss of growth and of growth we and the FGFR2 IIIb C1, C2, or C3 we the of the three FGFR2 IIIb variants to loss of growth by the of of in with the or the C1 variant activity. we found that the C2 variant the C3 variant caused activity than the C2 variant in (Fig. (Fig. we the of three FGFR2 IIIb variants to growth by with their the C3 variant more than the C2 and the C2 variant more than the C1 variant in (Fig. (Fig. of transforming by the FGFR2 IIIb isoforms in human not data that carboxyl-terminal sequences function of FGFR2 IIIb transforming with the loss of residues the causing the activation of transforming activity. of Y770F FGFR2 IIIb C1 in we determined the that to the enhanced transforming activity of FGFR2 IIIb variants carboxyl-terminal The carboxyl-terminal domain tyrosine residues of are of and for their cytoplasmic and growth regulatory The C3 variant lacks five tyrosine residues and the C2 variant lacks two tyrosine residues and present in the C1 (Fig. 1A). We hypothesized that the loss of tyrosine for the enhanced transforming activity of the C2 and C3 To we of the five tyrosine residues and of the carboxyl of the transforming C1 Because loss of tyrosine residues required for enhanced transforming we also generated and We of that wild and FGFR2 IIIb C1 proteins and their transforming in We found that the Y770F mutation alone in with the or mutations the activity the FGFR2 IIIb C1 receptor (Fig. and we found that the or the transforming FGFR2 IIIb C1 (Fig. the and that the Y770F mutation in to the the Y770F single (Fig. We found that FGFR2 IIIb C1 a FGFR2 IIIb C1 (Fig. that the in transforming activity not of in of protein Because the a carboxyl-terminal that is in C2 and C3, we not the that transforming of the C2 and C3 may in to of an of receptor we that of the the C1, C2, and C3 variants of FRS2 (Fig. suggesting that the three isoforms that loss of the Tyr-770 but not tyrosine residues and to the increased transforming of the C3 the C2 variant Tyr-770 (Fig. the loss of is not the for the increased transforming activity of the C2 is that the C2 variant lacks carboxyl-terminal residues found in the C1 but is also the C1 variant for three and (Fig. 1A). C1 C2 we that in three to the increased transforming of the C2 To we generated a of C1 that C2 but the (designated C2 than the We found that C2 a transforming the C2 variant (Fig. that the loss of the carboxyl-terminal to and not in and residues, is for increased transforming of the C2 of Y770F FGFR2 IIIb C1 in to in and J. PubMed Scopus Google that mutation of Tyr-770 alone not to enhance FGFR2 IIIb C1 growth transforming activity in for their is that but not the for FGFR2 IIIb D. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, R. Dickson C. PubMed Scopus Google Scholar). FGFR2 IIIb in but not by of an D. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, D. PubMed Scopus Google Scholar, J. A. J. PubMed Scopus Google Scholar). we the Y770F mutation enhanced FGFR2 IIIb C1 transforming activity in with the J. PubMed Scopus Google we found that the Y770F mutation not FGFR2 IIIb activity in (Fig. To the that expression of proteins to transforming we determined FGFR2 IIIb C1 a FGFR2 IIIb C1 (Fig. FGFR2 IIIb in to FGFR2 IIIb by R. Dickson C. PubMed Scopus Google Scholar). we determined the by enhance the transforming of the Y770F in To fibroblast and to the or Y770F fibroblast not enhance the activity of and the activity of the Y770F (Fig. that both loss of Tyr-770 and by are required for the enhanced transforming activity of the Y770F receptor in also a for the of and J. PubMed Scopus Google in To the of for the we used cell the to FGFR2 IIIb may function in to FGFR2 IIIb by not FGFR2 IIIb of receptor function not by activity by receptor activity. Leu-773 but Tyr-770 of the 770YXXL for FGFR2 IIIb C1 and we to mutation of the Tyr-770 enhanced FGFR2 IIIb C3 transforming activity. Interestingly, the Tyr-770 and flanking sequences to a putative motif that is to the major for of transmembrane proteins Rev. PubMed Scopus Google Scholar). a found that a Y770F mutation not FGFR2 IIIb M. 2005; PubMed Scopus Google Scholar). Because is that the tyrosine of the sorting motif with or J. Full Text PDF PubMed Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 2005; Full Text Full Text PDF PubMed Scopus Google we that Tyr-770 is with an of the sorting is also that both the Tyr-770 and Leu-773 residues of 770YXXL motif required for FGFR2 internalization and that concurrent mutation of both residues required to To the Tyr-770 Leu-773 residues are required for FGFR2 IIIb C1 we generated sequences four FGFR2 IIIb C1 and Because protein may not the of receptor internalization and we the of and to that or FGFR2 IIIb C1 proteins with or and with to the of the and four found with the in the intracellular (Fig. receptor with the to (Fig. to the the of the Y770F receptor also to a three the L773A and impaired to and (Fig. To FGFR2 IIIb we cell FGFR2 IIIb with a and the of of the FGFR2 IIIb to intracellular with we found that the Y770F mutation not receptor three the L773A and caused of internalization with the and Y770F (Fig. data that the Leu-773 but not Tyr-770 is for FGFR2 IIIb C1 to the and are Rev. PubMed Scopus Google Scholar). we determined the of Tyr-770 and Leu-773 residues in receptor that or YXXL with and the of FGFR2 IIIb protein for (Fig. caused a in both and Y770F in receptor protein with the of the three the L773A and a of in receptor protein with the of data that the Leu-773 but not Tyr-770 is for FGFR2 IIIb C1 of the Leu but Tyr of the 770YXXL of FGFR2 the in receptor internalization and correlated with aberrant activation of FGFR2 IIIb, that or and with or for 20 and with FGFR2 by with to FGFR2 IIIb caused increased tyrosine phosphorylation of the and four (Fig. the of both the and Y770F of tyrosine three internalization the Leu-773 and higher of FGFR2 IIIb with the and Y770F (Fig. data that loss of the Leu-773 in activation of FGFR2 IIIb that may caused by receptor internalization and of the Tyr and Leu of the 770YXXL FGFR2 IIIb by determined loss of the Leu-773 to cause impaired receptor and FGFR2 IIIb activation (Fig. also with an enhanced FGFR2 IIIb C1 transforming activity. We found that the L773A mutation alone to enhance the growth of FGFR2 IIIb (Fig. Because the Y770F mutation not receptor and not FGFR2 IIIb activation (Fig. with enhanced FGFR2 IIIb transforming activity (Fig. the Y770F and L773A mutations to cause distinct receptor function that we that the concurrent mutation of the Tyr-770 and Leu-773 residues cause of FGFR2 IIIb transforming activity of FGFR2 IIIb we found that both the and more than higher transforming activity with the Y770F more than or L773A more than single (Fig. To the that expression of proteins have an transforming we determined FGFR2 IIIb proteins (Fig. data that concurrent loss of the Tyr and Leu residues of the 770YXXL motif to enhance the transforming activity of the FGFR2 IIIb C3 of Tyr-770 FGFR2 IIIb C1 of the Y770F mutation not receptor (Fig. the Y770F mutation enhance the transforming activity of FGFR2 IIIb (Fig. that to the enhanced transforming activity of the Y770F that a Y770F of FGFR2 IIIb C1 and M. 2005; PubMed Scopus Google Scholar). in to found that the Y770F mutation than activity M. 2005; PubMed Scopus Google Scholar). To we to more the of Tyr-770 in FGFR2 IIIb regulation of and transforming activity. we determined mutation of Tyr-770 and 770YXXL motif residues caused tyrosine phosphorylation of and FGFR2 IIIb C1 and the C2 and C3 with and the of tyrosine phosphorylation the FGFR2 IIIb C1 and C2 variants that and not the C3 that lacks increased of (Fig. we found that three FGFR2 IIIb C1 that the Tyr-770 and to PLCγ, the L773A the to tyrosine phosphorylation of (Fig. the Tyr-770 is required for not determined by the M. 2005; PubMed Scopus Google Scholar). We found that in the wild and not Y770F FGFR2 IIIb C1 receptor (Fig. data that Tyr-770 is required for activation of by FGFR2 IIIb, and the loss of to to the enhanced transforming activity of the C3 variant that lacks of Tyr-770 of FRS2 but or is to and growth Google Scholar, A. J. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the that the Y770F mutation enhances FGFR2 IIIb C1 transforming in the loss of that mutation of Tyr-770 may activities that growth for is that loss of binding caused by the Y770F mutation may the activation of FGFR2 IIIb PLCγ, the FRS2 proteins the of (1Dailey L. Ambrosetti D. Mansukhani A. Basilico C. Cytokine Growth Factor Rev. 2005; 16: 233-247Crossref PubMed Scopus (530) Google Scholar, 2Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1495) Google Scholar, 3Grose R. Dickson C. Cytokine Growth Factor Rev. 2005; 16: 179-186Crossref PubMed Scopus (334) Google Scholar, 4Mohammadi M. Olsen S.K. Ibrahimi O.A. Cytokine Growth Factor Rev. 2005; 16: 107-137Crossref PubMed Scopus (557) Google Scholar). To loss of Tyr-770 altered FRS2 or of FGFR2 IIIb C1 the C2 and C3 variants and with by We found that with the Tyr-770 mutation and FRS2 tyrosine phosphorylation in the of (Fig. with FRS2 tyrosine phosphorylation also for the C3 that lacks Tyr-770 but not the C2 that Tyr-770 (Fig. The activation of FRS2 in the of to caused by receptor by by with for 20 three FGFR2 IIIb splice variants the of C1 increased of FRS2 (Fig. that the receptor variants to Because we FRS2 activation in Tyr-770 in the (Fig. we determined with activation of by We first loss of Tyr-770 to activation of and the we with a or with for 20 in and to a caused phosphorylation of in and to a the of of the FGFR2 IIIb proteins caused an in the of or (Fig. we determined mutation of Tyr-770 to activation and the and with by with to activity. we with a that the and of to activity. for 20 caused phosphorylation in and to a also a of phosphorylation in and in the of increased activation of or for or (Fig. data that loss of Tyr-770 caused FRS2 not activation of the or we found that loss of Tyr-770 not cause an in activation of the or is that both are but by present persistent signaling. we determined the activities of two important for Y770F transforming activity. We found that with the or the the growth of Y770F (Fig. that both and activities to the enhanced transforming activity caused by the Y770F mutation. of Tyr-770 FRS2 to we to loss of the to tyrosine phosphorylation of for is that binding to FGFR2 may with FRS2 and the Y770F mutation for FRS2 and binding to the loss of binding by the Y770F mutation may enhance FRS2 activity. To we determined the loss of the enhances FRS2 with the or of FGFR2 IIIb C1 and and with by N. Schlessinger J. Lax I. 20: PubMed Scopus Google that with FRS2 of receptor activation. with we also found that FRS2 with the wild and of FGFR2 we of FRS2 binding to FGFR2 in Tyr-770 with the or L773A (Fig. data that loss of Tyr-770 but not Leu-773 enhances FRS2 with and enhanced FRS2 with FGFR2 may to receptor activation of FRS2 for FRS2 and by the Y770F we determined FRS2 binding is required for the increased tyrosine phosphorylation and transforming activity caused by the Y770F mutation. we generated of FGFR2 IIIb C1 that that FRS2 to the of N. Schlessinger J. Lax I. 20: PubMed Scopus Google Scholar). also determined that mutation of the and residues in the and the FRS2 N. Schlessinger J. Lax I. 20: PubMed Scopus Google Scholar). Because the and residues in the of are in FGFR2 IIIb, to generate a we a of FGFR2 IIIb designated that is to the To FRS2 binding is required for activation of FRS2 by Y770F we mutations in the FRS2 binding in the Tyr-770 (designated WT, or and with and by with in we found that the Y770F mutation enhanced FRS2 tyrosine phosphorylation in the of and activation by the loss of FRS2 binding (Fig. that FRS2 binding to FGFR2 is for activation of FRS2 by the Y770F we determined loss of FRS2 binding and activation with a reduction in the transforming activity of the Y770F we the mutation the Y770F the a transforming activity with C1 (Fig. that the enhanced transforming caused by the Y770F mutation is FRS2 binding and activity. mutations of FGFR2 are found in human M. PubMed Scopus Google mechanism of FGFR2 activation in may alternative gene splicing and enhanced expression of the structurally and distinct C2 and C3 variant we the transforming of the C1, C2, and C3 splicing variants of FGFR2 IIIb and found that progressive loss of carboxyl-terminal sequences is correlated with increased transforming potency. Our mutation of the 770YXXL motif that the of motif to the highly transforming of the C3 splice variant by receptor internalization and increased receptor expression activation and by enhanced activation of FRS2 and the preferential expression of the C3 in mechanism for altered receptor that to of mutation of the YXXL motif FGFR2 IIIb C1 transforming determined internalization and expression and determined determined determined determined determined determined determined determined determined and in a of by and PubMed Scopus Google to the and transforming function of the FGFR2 IIIb splice variants in in are with that cause persistent of FGFR2 IIIb in we that a more for FGFR2 IIIb function in fibroblast where fibroblast a persistent of FGFR2 IIIb. found in FGFR2 IIIb and transforming activity of or in and fibroblast the of of FGFR2 IIIb function in with to FGFR2 IIIb function in in We determined that loss of the Tyr-770 alone enhanced the transforming activity of FGFR2 IIIb the Y770F is a and is a Because also to a putative YXXL sorting motif, we the of mutation of the 770YXXL motif receptor Surprisingly, we found that loss of Leu-773 but not Tyr-770 impaired receptor internalization and Interestingly, of receptor internalization correlated with activation of FGFR2 IIIb. We also found that concurrent mutation of the Tyr-770 and Leu-773 residues enhanced transforming activity. Furthermore, we found that loss of the caused FRS2 activation by enhancing FRS2 with of the impaired FGFR2 IIIb transforming a for FRS2 in FGFR2 IIIb receptor activation by loss of the 770YXXL support a where the enhanced transforming activity of FGFR2 IIIb C3 is of two distinct caused by the deletion of sequences that the 770YXXL motif, altered receptor and (Fig. to a found that a Y770F mutation than FGFR2 cell M. 2005; PubMed Scopus Google Scholar). are more with where activation been to than of to growth of and PubMed Scopus Google Scholar, M. J. Full Text PDF PubMed Google Scholar). the mutation in of and the to activation and their transforming activities in J. N. 2005; PubMed Scopus Google Scholar). for growth factor receptor activation of PLCγ, where loss of activation in loss of transforming activity A. J. Full Text Full Text PDF PubMed Scopus Google Scholar). in to that the mutation in activation but not by in M. N. M. Schlessinger J. PubMed Scopus Google Scholar, J. J. M. D. PubMed Scopus Google suggesting that activity is not required for growth for regulation of cell may distinct of may cell in function the for is that tyrosine may that cell that the a binding for that L. M. M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). to where we found that a Y770F mutation caused FRS2 tyrosine M. 2005; PubMed Scopus Google found that mutation FRS2 tyrosine We that may the of the the of of FGFR2 by support of of FGFR2 IIIb activation of FRS2 found in persistent is that the of and Full Text PDF PubMed Scopus Google Scholar, J. Trends Full Text Full Text PDF PubMed Scopus Google Scholar, 16: Full Text Full Text PDF PubMed Scopus Google Scholar). we found that increased FRS2 tyrosine phosphorylation in and Y770F to a increased FRS2 tyrosine phosphorylation in the Y770F but not receptor (Fig. the Y770F receptor activation of FRS2, and we that loss of FRS2 binding impaired we to that increased FRS2 activation not with increased activation of the or (Fig. is not FRS2 is also to regulatory to V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1495) Google Scholar, J. 2004; PubMed Scopus Google Scholar). of in phosphorylation of FRS2 residues, to of activation I. A. A. A. J. Schlessinger J. Full Text Full Text PDF PubMed Scopus Google Scholar). of a and activation of ERK, and of phosphorylation of FRS2 in activation I. A. A. A. J. Schlessinger J. Full Text Full Text PDF PubMed Scopus Google Scholar). that of or activation also Y770F receptor transforming activity that two FRS2 to that growth may activities that to identified. a for the transforming activity of the C3 of FGFR2 IIIb that two distinct by the 770YXXL the higher transforming activity with the the C3 variant more transforming than the for the enhanced transforming of the C3 we found that the C2 variant more transforming than the C1 the loss of the 770YXXL motif not to for C2 transforming and we have not found a for the enhanced transforming of the C2 FRS2 tyrosine phosphorylation and with proteins that may to and that the of the proteins important for growth We and of and for in and