Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

FRS2

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_356

Synonyms

Historical Background

FRS2 stands for fibroblast growth factor (FGF) receptor substrate 2. The FGF receptor substrate 1 is phospholipase (PLC) γ, which was identified as the first substrate of FGF receptor tyrosine kinases (Gotoh et al. 2004). Historically, SNT was identified as a protein that is tyrosine phosphorylated in response to nerve growth factor (NGF) or  FGF in PC12 cells and can bind to p13suc1, a yeast cyclin–dependent kinase binding protein, immobilized on agarose (Rabin et al. 1993). It was reported that there is a good correlation between the tyrosine phosphorylation of SNT and differentiation of PC12 cells. Then FRS2 was purified and cloned as a novel protein that is tyrosine phosphorylated in NIH3T3 cells in response to FGF (Kouhara et al. 1997). It turned out to be the same protein as SNT. Another protein that has homology with FRS2 was found in one of the expression sequence tag (EST) fragments. After the full-length cDNA of this protein was cloned, it was named as FRS2ß and the original FRS2 was renamed as FRS2α {Gotoh 2004 #1}. Other groups also identified this protein and named it SNT-2 or FRS3 (Xu et al. 1998; McDougall et al. 2001).

FRS2 Family Proteins Belong to a Group of Membrane-Linked Docking/Adaptor Proteins(MLDP)

Signal transduction pathways through receptor tyrosine kinases (RTKs) play pivotal roles in a number of aspects of physiological and pathological biology including cancer (Lemmon and Schlessinger 2010). Ample amounts of evidence indicate that proteins lacking catalytic activity, so-called scaffolding adaptor proteins, relay many key events of signal transduction from upstream components such as receptors to downstream elements (Pawson 2007). The scaffolding adaptor proteins that are upstream of RTK signal transduction are classified into two groups. One is comprised of docking proteins that have multiple tyrosine phosphorylation sites to dock downstream signaling proteins. They also often have other domains to bind other molecules. This group includes FRS2-, a new SH2-containing sequence (Shc), Grb2-associated binder (Gab)-, insulin receptor substrate (IRS)-, downstream of kinase (Dok)-family proteins-, and transmembrane adaptor proteins (TRAP). Several proteins in this group are subdivided into a group of membrane-linked docking protein (MLDP) (Fig. 1) (Gotoh and Tsuchida 2008). MLDP is localized in the lipid component of plasma membrane, since the protein contains membrane-anchor domain of a stretch of hydrophobic amino acid residues, pleckstrin homology (PH) domain, or transmembrane domain at or close to N-terminus. The other is comprised of adaptor proteins in a narrow sense. They have only SH3 or/and SH2 domains to bind signaling proteins. This group includes Grb2, Crk, and Nck.
FRS2, Fig. 1

Model of signaling through membrane-linked docking protein (MLDP). Schematic structure of several members of MLDPs. The phosphotyrosine binding domain (PTB) binds to phosphrylated tyrosine residues on receptors or signaling proteins. The pleckstrin homology (PH) domain binds to phospholipids such as phosphatidyl inositol (PI)-3 phosphate. The proline-rich domains are important for binding to SH3 containing proteins such as Src family tyrosine kinases or Grb2. Y designates potential tyrosine (Y) phosphorylation sites. The ERK binding domain in FRS2ß, Met binding domain in Gab1, or insulin receptor binding domain in IRS2 contains a unique sequence for binding to ERK, Met or insuling receptor

Some of the scaffolding adaptor proteins have phosphotyrosine binding (PTB) domain or Src homology (SH)2 domain to bind specific residues containing a tyrosine residue that becomes phosphorylated by activated RTKs or other tyrosine kinases. All the scaffolding adaptor proteins act in the specification and/or amplification of the signal transduction pathway. Since these scaffolding proteins were first discovered almost two decades ago, signal transduction pathways have emerged as a very complex network. FRS2 proteins are typical scaffolding adaptor proteins.

Domain Structure of FRS2 Proteins

FRS2α and FRS2ß are similar in structure. Both proteins contain amino acid residues MGSCCS, a consensus myristylation sequence (MGXXXS/T), at the N-terminus for binding to lipids in the membrane structure, including plasma membrane, constitutively (Fig. 2). Each has a PTB domain and multiple tyrosine phosphorylation sites at the C-terminus. FRS2α has six tyrosine phosphorylation sites and FRS2ß has five. The PTB domains are highly homologous and 72% of amino acids are identical. The residues surrounding each tyrosine phosphorylation site are similar but more than ten amino acids from the sites there is no similarity. Both proteins have binding sites for ERK; arginine residues (R) are critical for ERK binding to FRS2ß.
FRS2, Fig. 2

Schematic structures of FRS2 family proteins. The PTB domains of FRS2a and FRS2ß have 72% identity and 93% similarity in amino acids. Tyrosine-phosphorylated Grb2 binding sites activate Ras/ERK, ubiquination/degradation, and PI-3 kinase pathways. Tyrosine-phosphorylated Shp2 binding sites activate Ras/ERK pathway stronger than the Grb2 binding site. Arginine residues (R) in the ERK binding domain of FRS2ß are essetinal for binding to ERK

Molecular Functions of FRS2a

FRS2α proteins act as docking proteins downstream in certain types of RTKs, including FGF receptors (FGFR)s, neurotrophin receptors (TrkA, TrkB, TrkC), Eph (EphA4), RET, and ALK (Gotoh 2008). In particular, emerging evidence indicates that FRS2α acts as a central mediator for intracellular signaling in response to FGF, as described in detail below. FRS2α proteins bind to these RTKs via the PTB domain and become phosphorylated on tyrosine residues upon activation of these RTKs. In contrast, FRS2α proteins are not good substrates for other RTKs, insulin receptors, platelet-derived growth factor receptors, epidermal growth factor receptors (EGFR), and so on. Selectivity, in that FRS2α proteins choose only some tyrosine kinases for phosphorylation, is characteristic of this protein family.

The PTB domain is able to bind to specific peptides with or without tyrosine-phosphorylated residues. FRS2 proteins bind to non-phosphorylated peptides at the juxtamembrane domain of the FGFR via their PTB domains independently on ligand binding and receptor activation (Fig. 3). In contrast, binding between the PTB domain of FRS2α, TrkA and TrkB, or RET is mediated by tyrosine-phosphorylated peptides that have NPXY motif (X is any amino acid reside and Y is phosphorylated) in the receptors and is dependent on activation of the RTKs.
FRS2, Fig. 3

Binding sites of FRS2 proteins in RTKs. FRS2 binds to unphosphorylated peptides in the juxamembrane domain of FGF receptor. It binds to tyrosine-phosphorylated peptides in RET and TrkA and the binding sites are shared with those of other PTB domain-containing signaling proteins

Molecular functions of FRS2α are well studied in FGF signaling (Fig. 2) (Lemmon and Schlessinger 2010; Gotoh 2008; Eswarakumar et al. 2005). When FGFR is activated, FRS2α becomes tyrosine phosphorylated and creates two specific binding sites for Shp2, the SH2-containing tyrosine phosphatase, and four binding sites for the SH2 domain of Grb2. Several proteins are constitutively bound via two SH3 domains of Grb2: SOS, Cbl and Gab1. SOS is a guanine nucleotide exchange factor for Ras, and FRS2α-mediated recruitment of Grb2-SOS induces activation of the Ras/extracellular signal-regulated kinase (ERK) pathway. Cbl functions as an E3 ubiquitin ligase and the ternary complex FRS2α-Grb2-Cbl results in ubiquitination and degradation of FRS2α and its receptors. This is one of the negative feedback mechanisms of FGF signaling. The ternary complex FRS2α-Grb2- Gab1 enables tyrosine phosphorylation of Gab1 followed by recruitment of phosphatidyl inositol (PI)-3 kinase and activation of a cell survival pathway. SOS binds to the N-terminal SH3 domain of Grb2, Gab1 binds to the C-terminal SH3 domain of Grb2, and Cbl binds to both N- and C-terminal SH3 domains of Grb2. Thus, FRS2α assembles both positive and negative signaling proteins to mediate a balanced signal transduction through Grb2. The FRS2α-Shp2 complex induces tyrosine phosphorylation of Shp2 followed by strong activation of ERK in response to FGF. Tyrosine-phosphorylated Shp2 activates its own tyrosine phosphatase, resulting in strong activation of the Ras/ERK pathway in numerous cell contexts. The Grb2-binding sites of FRS2a activate ERK at more moderate levels.

Signaling pathways activated through Trks and RET downstream of FRS2α upon activation of these RTKs appear to be similar to FGF signaling. Tyrosine phosphorylation of FRS2α is also induced by the activation of EphA4. FGFRs and EphA4 form complex proteins by direct interactions between the juxtamembrane domain of FGFRs and the N-terminal portion of the tyrosine kinase domain of EphA4. Activation of EphA4 leads to the activation of FGFRs and tyrosine phosphorylation of FRS2α.

FRS2α is phosphorylated by ERK at multiple threonine residues in response to a variety of ligands, FGF, insulin, EGF, and PDGF. These include extracellular stimuli that do not induce tyrosine phosphorylation of FRS2α. There are eight canonical ERK phosphorylation sites (PXTP motif) in FRS2α. In addition, activated ERK binds to threonine phosphorylated FRS2α (Fig. 2). Thus there is an ERK-mediated negative feedback mechanism for the control of signaling pathways that are dependent on FRS2α.

Physiological Roles of FRS2α

The expression of Frs2α can be detected at embryonic day (E) 5.5 in mouse embryos and is expressed ubiquitously during development. FRS2α play critical roles for a variety of FGF-induced developmental processes and maintenance of tissue type–specific stem cells that are dependent on FGF. Several Frs2α mutant mice were generated: Frs2α-knockout mice and two knock-in mice that express a mutant form of FRS2α whose tyrosine residues of Grb2 (Frs2α 4F mutant)- or Shp2 (Frs2α 2F mutant)-binding sites are replaced with phenylalanine (Fig. 4) (Gotoh 2008). Embryos of the Frs2α-knockout mouse have defects in the FGF4-dependent maintenance of trophoblast stem cells and show developmental retardation, resulting in embryonic lethality by E8 (Gotoh et al. 2005; Murohashi et al. 2010). Frs2α 2F/2F mice display multiple developmental defects and show perinatal death, though Frs2α 4F/4F mice can survive as adults and show no gross morphological defects except an eyelid developmental defects. Embryos of Frs2α 2F/2F mice lack carotid body, have defective eye development, and show anophthalmia (no eyes) or microphthalmia (small eyes). Neural progenitor cells of Frs2α 2F/2F mice show reduced proliferation in response to FGF stimulation. FRS2α is also essential for self-renewing activity of neural stem/progenitor cells (NSPCs) in response to FGF (Sato et al. 2010). Specific ablations of Frs2α in the prostate epithelium inhibit prostatic branching morphogenesis and growth (Zhang et al. 2008).
FRS2, Fig. 4

FRS2α is a central mediator in FGF signaling in vivo. Frs2α null mouse embryos showed early embryonic lethality due to a failure of maintenance of trophoblast stem (TS) cells. TS cells are dependent on FGF4 and localized in extraembryonic ectoderm (ExE). The wild type ExE is positive in pERK staining but it is weak in the mutant. The Frs2α 2F/2F mice have a variety of developmental defects and some of them are reasonably explained by failure of FGF signaling

Molecular Functions of FRS2ß

FRS2ß binds to a few RTKs including FGFR, Trk, and ALK through the PTB domain and becomes phosphorylated on tyrosine residue, similar to FRS2α. Like FRS2α, Grb2 and Shp2 are recruited to phosphorylated tyrosine residues on FRS2ß upon activation of RTKs, leading to the activation of ERK (Fig. 2). FRS2ß also binds to EGFR family members, including EGFR and ErbB2, constitutively with its PTB domain regardless of the absence or presence of ligands (Fig. 5). However, FRS2ß is not phosphorylated by EGFR RTK in this case. Instead, FRS2ß inhibits the activity of EGFR. Expression of FRS2ß inhibits EGF-induced autophosphorylation of EGFR, and also decreases activation of its downstream signaling proteins. The binding between FRS2ß and phosphorylated ERK is important for inhibition of EGFR signaling. This forms a negative feedback loop after the activation of ERK downstream of the activated EGFR, or to maintain the EGFR in an inactive state after the activation of ERK by other stimuli. Therefore, it appears that FRS2ß is a unique scaffolding adaptor that serves as both a negative and a positive regulator for receptor species–dependent RTK signaling (Iejima et al. 2010).
FRS2, Fig. 5

FRS2ß constitutively binds to EGFR family members. Activated EGFR family members activate the ERK pathway. The activated ERK binds to FRS2ß and inhibits the EGFR signalling

Physiological Roles of FRS2ß

In contrast to the rather ubiquitous expression pattern of Frs2α, Frs2ß is expressed only in a limited number of tissues, including central nervous systems (CNS) and other epithelial tissues, such as lung epithelial cells and the gastrointestinal tract (Gotoh et al. 2004; Minegishi et al. 2009). Though both Frs2α and Frs2ß are expressed in CNS, the expression pattern of these genes tends to be mutually exclusive. For example, though Frs2α is strongly expressed in the ventricular zone of mesencephalon, expression of Frs2ß is weak there. The distinct expression patterns of Frs2α and Frs2ß suggest the possibility that they have different physiological roles. In contrast to the well-known functions of FRS2α, the functions of FRS2ß have long been unclear.

Relevance of FRS2 Proteins to Cancer

It is known that FGF ligands and their recepors are overexpressed in a variety of cancers, including of the breast, stomach, prostate, pancreas, bladder, and colon (Sato and Gotoh 2009). Activating mutations in FGFR3 are observed frequently in bladder and cervical carcinoma and in some cases of multiple myeloma, while activating mutations in FGFR2 are found in gastric carcinoma. Further, several types of fusion proteins involving FGFR1 caused by chromosomal translocation are found in hematologic malignancies. Given that aberrant FGF signaling is important in tumorigenesis based on all these reports, it is reasonable to predict that FRS2 proteins are also involved in tumorigenesis.

Somatic rearrangements of the TrkA gene are detected in a fraction of papillary thyroid carcinomas and produce chimeric proteins. All of them contain a portion of TrkA, including tyrosine kinase domain. The resultant thyroid Trk oncoproteins activate FRS2α and FRS2ß. Gene rearrangements leading to fusion of the kinase domain of RET with heterologous proteins containing dimerization motifs result in constitutively activated RET proteins. Such fusion proteins are expressed in some cases of papillary thyroid carcinoma. Germ line point mutation in RET results in inherited multiple endocrine neoplasm types 2A and 2B and familial medullary thyroid carcinoma. FRS2α is tyrosine phosphorylated by ligand-stimulated and constitutively activated oncogenic forms of RET, leading to activation of ERK. The NPM-ALK oncoprotein activates FRS2α and FRS2ß and transforms NIH3T3 cells.

Expression of FRS2ß suppresses EGF-induced cell transformation and proliferation. High expression levels of FRS2ß significantly correlate with good prognosis of non-small lung cancer patients (Iejima et al. 2010). These findings suggest that FRS2ß is suppressive for tumorigenesis in which aberrant EGF signaling is involved.

Summary

There are two members – FRS2α and FRS2ß – in the FRS2 family of docking/scaffolding adaptor proteins. These proteins function downstream of certain kinds of RTKs that are important for tumorigenesis, including the FGFR, Trk, RET, and ALK. Activation of these RTKs allows FRS2 proteins to become phosphorylated of tyrosine residues and then bind to Grb2 and Shp2, a SH2 domain-containing adaptor and a tyrosine phosphatase, respectively. Subsequently, Shp2 activates a Ras/ERK pathway and Grb2 activates a Ras/ERK, PI-3 kinase, and ubiquitination/degradation pathways by binding to SOS, Gab1, and Cbl via the SH3 domains of Grb2. FRS2α acts as a central mediator in FGF signaling. FRS2α mutant mice exhibit a variety of defects in developmental processes and in maintenance of tissue type–specific stem cells, such as trophoblast stem cells and neural stem cells; many of them are due to defects in FGF signaling. Although FRS2ß binds to EGFR family RTKs, including EGFR and ErbB2, it does not become tyrosine phosphorylated. Instead, it inhibits EGF signaling, resulting in inhibition of EGF-induced cell proliferation and cell transformation. FRS2ß may have a tumor suppressive role for non-small cell lung cancer.

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Institute of Medical ScienceThe University of Tokyo, Minato-kuTokyoJapan