Growth Factor Receptor-Bound Protein 2
Created By: Robert Denney
Growth factor receptor-bound protein 2 (PDB ID: 2H5K) in complex with a Shc-derived ligand serves an essential function in multiple cell signaling pathways that mediate cell growth and differentiation1,2. Grb2 functions as an adaptor protein that consists of a single SH2 domain flanked by SH3 domains on the N and C terminal sides. The Sh2 domain is separated from the Sh3 domains with long flexible “arms” also known as linkers. It consists of 217 amino acids and is 25 kDa3. It can form as a dimer with 2 chains, or function as a monomer, however, the crystal structure examined here forms as a dimer. The SH2 domain binds phosphotyrosine residues on Shc-derived ligands or growth factors, while the SH3 domain facilitates association with additional cell signaling proteins1. This protein was elucidated in a study in rats that determined it served to link receptor tyrosine kinases to Ras signaling3.
One of the most studied and important pathways in cell signaling involves the activation of Ras, and the interaction of Ras with mitogen-activated protein kinases (MAPK) to begin a cell signaling cascade. The Ras signaling pathway recruits multiple transcription factors and molecules involved in cell differentiation and growth. On binding their respective growth factors or hormones, receptor tyrosine kinases function as self-acting enzymes phosphorylating its intracellular chains at tyrosine residues. The Sh2 domain of Grb2 (SRC homology domains) can associate with these phosphotyrosine residues, or an additional protein family known as Shc-derived ligands which also become tyrosine phosphorylated in response to growth factors. The next step in the pathway involves the SH3 domains of Grb2. These domains can associate by binding with a 10-residue proline rich amino acid sequence of Sos (Son of seveless) proteins. This protein is a guanine nucleotide exchange factor which can then associate with and activate Ras, resulting in the activation of the MAPK cascade, as well as phospholipase C and activation of a multitude of transcription factors that facilitate cell growth and differentiation, as well as cytoskeletal organization, survival, and calcium signalling2,6.
As stated above, the Sh2 domain binds primarily to a specific sequence that contains a phosphotyrosine residue. Cell receptors are often autophosphorylated at tyrosine residues on activation by ligand, and the Sh2 domain can bind these residues2. In Grb2, the Sh2 domain consists of a central antiparallel beta sheet which is flanked by two helices4. This subunit binds to phosphotyrosine residues surround by the specific amino acid sequence YxNx, with optimal binding to the sequence YVNV. This binding occurs between two primary surfaces of the SH2 domains which make up the overall binding pocket1,4. The first is a phosphotyrosine binding surface consisting of the residues Arg-67, Arg-86, Ser-88, Ser-90, His-107, and Lys-109, and the second is a binding surface for residues following the phosphorylated tyrosine residue. Within the first site, the binding pocket, the carbonyl oxygen atom of the phosphorylated tyrosine residue interacts with Arg-67, forming hydrogen bonds1. The phosphate is stabilized by hydrogen bonds with the basic amino acids above Arg-67 and Arg-86, as well as Ser-90 and Ser-96. Hydrogen bonding is necessary to stabilize the highly electronegative phosphate moiety.
The second surface is a hydrophobic surface responsible for specificity for the remaining amino acids in the YxNx sequence, and consists of the important residues Gln-106 and Phe-108, which participate in H-bonding with valine in the Shc-derived ligand, in this case, Acetyl pYVN1. Other important residues in this site include Leu-120, Trp-121, Tyr-134, and Leu-148. Asparagine of the peptide sequence interacts with the carbonyl oxygen of Lys-109 in the first binding pocket, helping to stabilize the sequence. Point mutations of Trp-121 to threonine reduced the affinity for peptide by approximately 14-fold. Evidence indicates that depending on the ligand binding as well as dimerization, it may take either an open or closed conformation with varying affinities1.
Grb 2 shows significant homology with many proteins, and is highly evolutionarily conserved. The modular construction of SH2 and SH3 domains likely enabled increasing complexity through combination different domains, rather than changes in the protein sequence itself, which could account for such high conservation of the protein. Of particular note is sequence homology with Sem-5 (PDB ID: 1KFZ), the nematode protein3,4. Despite the large differences between C. Elegans, and humans, Grb2 shows 63% sequence similarity to Sem-5 indicating a similar function. Studies in the nematode C. Elegans demonstrated that Sem-5 is also involved in a similar cell signaling process, and enables activation of Ras and cell growth. They have also helped to elucidate the structure of the
The Sh3 domain (PDB ID: 1IO6) of Grb2 forms a β-barrel, a highly conserved structure within the Sh3 family with the ability to bind to multiple different proline rich-motifs4. An example of a proline-rich motif the Sh3 domains of Grb2 could bind is the amino acid sequence VPVPPPVPPRRRP. These are most often found on proteins such as SOS, and other important signaling molecules. SOS in particular is involved in the Ras signaling pathway, and acts as a guanine nucleotide exchange factor to activate it4.
Sem-5, a homologue of Grb2, has been especially useful in elucidating the function and mechanisms of the Sh3 domain. Studies have shown that the N-terminal arginine has is an important residue in the proline-rich peptides that Grb2 binds. Point mutations in the homologue Sem-5 have been studied in C. Elegans and provide evidence for the function of several conserved residues including Glu-15 (in the C-terminal SH3 domain) and Glu-174 (in the N-terminal SH3 domain)3. As mentioned above, Sh3 domains such as those of Grb2 can bind the class II polyproline amino acid motif xPPxPPR, and interact with them in the formation of a polyproline II helix. On mutation of N-terminal arginine to lysine, binding of Grb2 to the ligand is greatly reduced. It was shown that this arginine guanidino group forms forms salt bridges with the Glu-16 and Glu-174 residues mentioned above. This interaction is conserved in both Sh3 domains of Grb24,5.
SH3 binding can occur in two different orientations depending on whether the arginine of the polyproline motif is N or C-terminal. Additional interactions between Trp-36 and Tyr-52 (N-Sh2) and Trp-193 and Tyr-209 (in C-SH2) may determine the shape and Shc-derived ligand affinity by interacting with other amino acids in the motif such as valine, which can pack against the conserved tyrosine and tryptophan residues mentioned above. It is most likely that both Sh3 domains participate during interactions of biological significance, such as the binding of Grb2 to SOS, a guanine nucleotide exchange factor4, 5.
The SH2 and SH3 domains of Grb2 work together in order to recruit signaling molecules to activated receptors on the cell membranes. The SH2 domain binds to the receptor, while the Sh3 domains bind Shc-derived ligands such as SOS, which continue the signaling cascade2. The structures of each domain, and the presence of two different types of domains allow Grb2 to fufill its role as a modular adaptor protein. The high sequence homology and critical role in cell signaling, differentiation and development make Grb2 and its Shc-derived ligands an extremely important field of study. Ras signaling has been implicated in cancers6, and understanding the components and functions Grb2 plays are particularly important in treating and understanding diseases that involve dysregulation of cell growth and signaling pathways. Additionally, the remarkably high conservation of Grb2 across is invaluable in studying evolution; homologues of Grb2 and other molecules involved in ras signaling are present in organisms as evolutionarily distant from humans as nematodes and can provide insight to apparent similarities in development, even in such distant organisms.