Allosteric regulation of GRB2 modulates RAS activation

ABSTRACT

RAS activation is a multiple‐step process in which linkage of the extracellular stimuli to the RAS activator SOS1 is the main step in RAS activation. GRB2 adaptor protein is the main modulator in SOS1 recruitment to the plasma membrane and its activation. This interaction is well studied but the exact mechanism of GRB2-SOS1 complex formation and SOS1 activation has yet remained obscure. Here, a new allosteric mechanism for the GRB2 regulation is described as a prerequisite for the modulation of SOS1 activation. This regulatory mechanism comprises a series of intramolecular interactions which are potentiated by GRB2 interaction with upstream ligands.

Abbreviations: GRB2, growth factor receptor-bound protein 2; SOS1, son of sevenless 1; RAS, Rat Sarcoma; GEF, guanine nucleotide exchange factor; GAP, GTPase‐activating protein; HER2, human epidermal growth factor receptor; SH3, SRC Homology 3; SH2, SRC Homology 2; PRD, proline-rich domain; PRM, proline-rich motif; PRP, proline-rich peptide; RTK, receptor tyrosine kinases.

KEYWORDS:

• Allosteric regulation
• GRB2
• GTPase
• SOS1
• RAS
• RASGEF

Nature has evolved sophisticated, cell-type-specific mechanisms to sense, amplify and integrate diverse external signals, and ultimately generate the appropriate cellular response. Signals are processed by evolutionarily conserved signalling cassettes that comprise specific constituent components acting as receptors, mediators, effectors, and regulatory proteins. GRB2, for instance, links activated receptor tyrosine kinases (RTKs) to the membrane-associated RAS by recruiting the RAS activator SOS1 to the plasma membrane. This action consequently promotes SOS1-mediated activation of RAS paralogs, e.g., the protooncogene KRAS4B, which in turn regulate various signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway [1].

The RTK-RAS-MAPK axis is a highly conserved, intracellular signalling pathway that plays an essential role throughout mammalian development, from embryogenesis to tissue-specific cellular homoeostasis in the adult [2]. Dysregulation of components or regulators of this cascade is frequently associated with tumour growth and a distinct subset of developmental disorders called the RAS-MAPK syndromes or RASopathies [3–5]. This signalling cascade has rapidly taken centre stage in cancer and RASopathy therapies [6,7].

Significant advances have been made in understanding the spatiotemporal features of the constituent members of the RTK-RAS-MAPK axis, which are linked and modulated by a large number of accessory proteins [8]. They are an essential family of proteins that fine-tune assembly and spatiotemporal organization of the constituent signalling components and maintain the specificity and function of signal transduction. [9]. They can be functionally divided into at least four subgroups: anchoring, docking, adaptor, and scaffold proteins.

One of the best-investigated accessory proteins is GRB2. It is an ubiquitously expressed and evolutionarily conserved adaptor protein [10] that links extracellular signals to a variety of pathways [10–12]. GRB2 was discovered as an epidermal growth factor receptor-binding protein with no intrinsic enzymatic activity [13,14]. It has been shown to be a positive regulator of RAS signalling crucial for embryogenesis as well as malignant transformation [13,15]. It is involved in T-cell antigen receptor (TCR) signalling through its association with LAT and CD3 complex in T cells [16].

GRB2 is a 25-kDa protein and consists of three protein interaction modules, one SRC homology 2 (SH2) and two SRC homology 3 (SH3) domains an N-terminal SH3 or nSH3 and a C-terminal SH3 or cSH3. The SH3 domains typically bind to the proline-rich motifs (PRMs) of target proteins, e.g., RAS-specific guanine nucleotide exchange factor (RASGEF) SOS1 and GRB2-associated-binding protein 1 (GAB1) [17–23]. The SH2 domain specifically binds to tyrosylphosphate (pY)-containing proteins, such as activated RTKs (e.g., HER2) and other transmembrane proteins, such as LAT [14,24–26]. The GRB2 SH2 domain has been shown to bind to a pY-Φ-N-Φ consensus motif (where Φ represents a hydrophobic residue) of activated receptors [26]. The SH3 domains of GRB2 exhibit different specificity for proline-rich containing proteins. nSH3 recognizes the consensus motif P-x-x-P-x-R and interact with SOS1, CBL, and dynamin [27,28], whereas cSH3 binds GAB1 and SLP-76 containing the consensus motif P-x-x-x-R-x-x-K-P [27].

Proline-rich domains (PRDs) consist of a high percentage of proline residues, ranging between 14% and 49% [29], available in distinct PRMs with a length of 10–25 amino acids. For instance, SOS1 PRD contains 21% proline residues (60 prolines in 284 amino acids), and human ALIX protein, another adaptor protein involved in membrane remodelling, even 30% [30]. Accordingly, a PRD is defined as a domain that consists of at least three PRMs.

The binding of the GRB2-SOS1 complex to the tyrosine phosphorylation sites on cell surface proteins has been proposed to translocate SOS1 to the plasma membrane in the vicinity of RAS. This enables GDP/GTP exchange on RAS, leading to activation of the mitogen-activated protein kinase (MAPK) cascade [31,32]. The GRB2-GAB1 complex is known to activate the protein tyrosine phosphatase SHP2. The GRB2-GAB1-SHP2 complex downregulates the PI3K pathway and induces RAS activation [33,34].

The interaction of GRB2 with SOS1 was first described in the early 1990s [35–37]. The first study by Lemmon et al. showed that GRB2 forms a 1:2 complex with a SOS1 peptide with a binding affinity of 22 µM, and a 1:1 complex with HER1 pYP, with a binding affinity of 0.4 µM [37]. This study and Cussac et al. proposed independent ligand binding to the SH2 and SH3 domains of GRB2 [37,38]. Since then, many different groups have investigated GRB2 interactions with various SOS1 peptides with nanomolar to millimolar binding affinities for GRB2 full-length (FL), nSH3, and cSH3 using different methods and varying conditions [20,39–45]. Moreover, GRB2 nSH3 has been generally appreciated as the main SOS1 binding module, and the cSH3 as a nodule that increases the overall stability of this protein complex. The GRB2-SOS1 interaction has been very recently proposed to induce a closed conformation in nSH3, while the cSH3 conformation remains unchanged [46]. McDonald et al. proposed the formation of the SOS1-GRB2-GAB1 complex [33]. Accordingly, SOS1 binding to nSH3 induces a conformational change in GRB2, allowing GAB1 to access the cSH3 domain in a non-competitive manner. This may mean that the association of one molecule of GRB2 with its upstream ligands, e.g., HER2 or LAT, reciprocally controls two distinct pathways, namely, the PI3K and MAPK pathways [31–34].

GRB2-SOS1 complex formation requires the interaction of GRB2 SH3 domains with the C-terminal PRD of SOS1 [36,47]. The SOS1 PRD contains more than 10 PRMs, most of which do not seem to bind GRB2 SH3 domains [42,43,45,48]. The best-investigated SOS1 PRM is VPVPPPVPPRRR (here called reference peptide 1 or RP1). RP1 has been reported to bind GRB2 nSH3 more tightly than cSH3 [20,38,45,49]. RP1 binding has been recently shown to induce a closed conformation of nSH3 [45]. The PRM-binding deficient variants of GRB2 SH3 domains (substitution of tryptophan 36 and/or 193 for lysine) have been shown to abolish the critical role of GRB2 in ERK activation via the SOS1-RAS-RAF-MEK axis [47,50,51].

These mutations disrupt nSH3/cSH3 binding to SOS1. Thus, the GRB2-SOS1 interaction is a key step towards proliferation in normal and cancer cells [52]. Targeting the GRB2− SOS1 interaction has been suggested to offer new avenues for future therapeutic strategies for upstream mutations in cancer, such as in EGFR [45].

Allosteric regulation of proteins is widespread in a variety of biological processes [53]. In multivalent proteins, the signal can be propagated from one site to other sites by allosteric mechanisms. Allosteric regulation of not only enzyme activities but also many scaffold proteins is a mechanism for fine-tuning the biochemical reaction pathways controlling the on/off states of the proteins [54,55]. A quantitative description of the communication between two distinct sites in a multivalent protein is still very challenging. In the case of SH3 domains of GRB2, while they have often been associated with allosteric mechanisms, the intradomain communication between residues has been poorly explored [56]. Kazemein Jasemi et al. have investigated in-depth GRB2 and its interaction with proline-rich peptides (PRPs) of SOS1 in the presence and absence of HER2 pY peptide (pYP) [48]. Therefore, a huge set of reagents were systematically used, including 50 different variants of GRB2 full-length, isolated SH2 and SH3 domains, deletion variants of the short linker segments of GRB2 interconnecting its globular domains (amino acids 54–63 and 149–159) as well as the four very C-terminal amino acids NRNV, 14 different peptides originating from HER2, SOS1 and the RHO GTPase WRCH2, and various methods to measure the interaction and determine the binding affinities. This cell-free study demonatrates that allostery is an intrinsic property of the GRB2 conformational ensemble, governed by upstream ligand binding, e.g., dimerized HER2 receptor, and essential for a stepwise SOS1 binding and activation. The results allowed to propose an allosteric modulation model for the adaptor protein function of GRB2 in linking the upstream signals with a SOS1-mediated activation of RAS as illustrated in Figure 1.

Figure 1. A proposed mechanism for the allosteric regulation of GRB2. Allosteric regulation of GRB2 is a multi-step process that appear to be a functional prerequisite for potential bivalent SOS1 binding. It possibly starts with the interaction of SOS1 proline-rich motif 3 (PRM 3) with GRB2 nSH3 domain (i). The cytosolic, heterodimeric, inactive GRB2-SOS1 complex translocates to dimerized, tyrosine-phosphorylated HER2 receptor, where pYP binding of the SH2 domain (ii) may induce its association with phospholipids of the plasma membrane (iii), as proposed by Park et al. [57]. This event may not only increase the dwell time of GRB2-SOS1 complex at the membrane but rather triggers the release of cSH3 from its interaction with SH2 (iv) and the subsequent back-to-back interaction of cSH3 with nSH3 (v). This cooperative mechanism now allows cSH3 to bind SOS1 PRM 4 (vi) and ultimately induces among other events the SOS1-catalysed RAS activation (vii).

Taken together, Kazemein Jasemi et al. have provided comprehensive data on the molecular interactions of PRDs, PRMs, and Y-/pY-peptides with GRB2 in an attempt to shed light on the mechanism underlying RTKs linking with SOS1 by GRB2 [48]. They demonstrate that the mechanism by which GRB2 functions as an adaptor protein is not based on a simple binding and recruitment model but also a multistep allosteric mechanism of SOS1 activation. GBR2 rather binds to three out of ten SOS1 PRMs and essentially utilizes both functional GRB2 SH3 domains for the interaction with SOS1. A central issue is a reciprocal relationship between the two SH3 domains that determine their successive interactions with SOS1, leveraged by a unique C-terminal NRNV motif. Thus, GRB2 appears to undergo, upon upstream ligand binding (e.g., HER2 pYP), a series of structural transitions from one site to a physically distinct site that may reinforce a stepwise association of downstream ligands (e.g., SOS1 PRMs). Such signal propagation is on the one hand achieved via both interdomain structural changes and allosteric networks induced by HER2 pYP binding to the SH2 domain and, on the other hand, modulated by specific domain–domain rearrangements, which ultimately results in the engagement of both SH3 domains in binding and eventual activation of SOS1.

Acknowledgments

We are grateful to our colleagues from the Institute of Biochemistry and Molecular Biology II of the Medical Faculty of the Heinrich-Heine University Düsseldorf for their support, helpful advices, and stimulating discussions.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the German Federal Ministry of Education and Research (BMBF) – German Network of RASopathy Research (Bundesministerium für Bildung und ForschungBundesministerium für Bildung und Forschung GeNeRARe; grant number: 01GM1902C), and the European Network on Noonan Syndrome and Related Disorders (NSEuroNet; grant number: 01GM1621B).