Grb10 SH2 Domain

From Proteopedia

Introduction Grb10 (Growth factor Receptor-Binding protein) is the member of a family of adapter proteins (Grb7 and Grb14) that interacts with tyrosine kinases. [1] This protein acts as a growth suppressor by competitively binding to insulin receptors and IGF1R (Insulin-like growth factor 1 receptor) which prevents other hormones to bind and facilitate chemical reactions within the cell. It has also been found to interact with E3 Ubiquitin Ligase NEDD4 which promotes the degradation of IGF1R. New developments in the research of Grb10 have shown that by inhibiting the expression of this gene, mice express a phenotype that has an increase in body mass, improved glucose homeostasis, improved insulin sensitivity and reduced adiposity. Grb10 knockout mice have a 30% increase in muscle mass, which has designated it to be called the 'Hulk' protein. Dimerization of the Grb10 SH2 Domain The crystal structure of Grb10 SH2 (Src Homology) domain (molecular mass = 12.4 kDa) was an important step to understanding how this protein interacts with IGF1 receptors, and although the SH2 domain functions as an independent segment, it forms a dimer in physiological environments. [1] The dimer interface exists due to the middle hydrophobic Phe515 and uncharged Thr504 (labeled blue) residues packed into its equivalent counter parter on the other protomer, designated as Phe515' and Thr504' (labeled red); Gln511 (blue) forms a hydrogen bond to the backbone of Asp514' (red) while the side chain of Asn519 forms two hydrogen bonds to the backbone of Lys505'. [1] The interface ends with Leu518 and Phe-496' via hydrophobic interactions. [1]. The structure of Grb10 SH2 forms similar SH2 domains found in other proteins, which have an alpha helix on the outsides with anti-parallel beta sheets. To ensure the crystallographic structure of Grb10 SH2 is indeed a dimer in solution, Evan Stein and colleagues substituted Phe515 at the dimer interface with arginine (electrically charged side chain) and found, using gel filtration chromatography (Picture 1), that the Grb10 SH2 dimer had indeed become independent monomers. Picture 1 Picture 1 The gel used for filtration chromatography had 5 lanes with 5 different components: BPS-SH2_WT (Wild Type) BPS-SH2_F515R (mutant = Phe515 --> Arg) IRK_3P (tris-phosphorylated Insulin Receptor Kinase domain) BPS-SH2_WT + IRK_3P BPS-SH2_F515R + IRK_3P As seen in lanes 1 and 2, the BPS-SH2 proteins did not travel down the gel due to their high pI; to resolve this issue, the researchers added IRK_3P to the two BPS-SH2 proteins which then made a complex that was mobile. [1] Lane 4 shows a band labeled 2:2 complex that shows the position of the SH2 dimer. The additional band found at the very top of lane 4 represents the BPS-SH2_WT protein that did not complex with high motility protein IRK_3P, i.e. it was not able to migrate through the gel due to its high pI. Lane 5 shows a band labeled 1:1 complex elucidating that the Arg substitution at Phe515 did indeed produce a monomer, which was able to travel farther down the gel. Why BPS-SH2? In order for the full-length Grb10 protein to interact with Insulin Receptor Kinase (IRK), the SH2 and BPS domain must be present. [2]

spinqualitylabelsall models Crystal Structure of the SH2 Domain of Grb10 (PDB entry 1nrv) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Interaction Between Grb10 and E3 Ubiquitin Ligase NEDD4

spinqualitylabelsall models Crystal structure of the Nedd4 C2/Grb10 SH2 complex PDB entry 3m7f) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Grb10 has now been shown to not only inhibit insulin receptors and IGF1R kinase activity, but also interacts via its SH2 domain with the C2 domain of E3 ubiquitin ligase NEDD4 facilitating ubiquitation of IGF1R ^[3]. It is hypothesized that the Grb10 SH2 interaction with the E3 domain of NEDD4 may allow NEDD4 to come in close proximity to IGF1R promoting degradation.^[4]

There are 3 interfaces at which Nedd4 C2 and Grb10 SH2 interact:

Interface I is the largest of the 3 interfaces between Grb10 SH2 and NEDD4 C2 domains. There are 8 hydrogen bonds formed at this interface (3 backbone H-bonds and 4 residue H-bonds), with the least important H bonds formed between ASN519 and LEU177, for disruption between these two residues with an induced mutation did not inhibit Grb10 SH2 and NEDD4 C2 to complex. ^[5]

Interface II, the smallest of the 3, is composed of residues 429-434 of Grb10 SH2 interacting with residues 112-114 of NEDD4 C2; disruption of this interaction does not disrupt the SH2-C2 domain complex. ^[6]

Interface III is made of NEDD4 C2's proline rich C terminus which complexes with Grb10 H2 N-terminal residues 431-440 and C-terminal residues 532-535; among this interaction ARG533 (SH2) and PRO284 (C2) forms a hydrogen bond and ARG431 (SH2) and PRO287 (C2) forms a slat bridge. ^[7]

Grb10 Gene Inhibition Affects Body Composition, and Insulin Signaling

Many studies have shown that when mice are subjected to Grb10 gene disruption (chromosome 11) during prenatal life, they become approximately 30% larger in muscle mass, have improved glucose homeostasis, improved insulin sensitivity and reduced adiposity, i.e. fat storage, during their postnatal life ^{[8] [9]}.

Picture 2

Picture 2 shows how inhibition of the Grb10 affects the overall growth of a mouse fetus. ^[10] In section a, it is shown that the wild-type and paternal Grb10 knockout mice have virtually the same weight, suggesting that it is not the paternal Grb10 allele that inhibits overally body mass. The middle bar represents the maternal knockout Grb10 mouse and shows an approximate 30% increase in overall weight when compare to wild-type. This data is visualized in section b, where the maternal Grb10 knockout mouse (left) sows a significant size difference when compared tot he wild-type mouse (right). Directly below section a, a bar graph representing the percent total body weight of specific vital organs. The liver shows an overall increase in percent body mass while the brain shows a slight decrease. More research must be done to determine why the brain and liver are specifically affected by Grb10 inhibition.

Medical Implications for Grb10 Inhibition

The Grb10 gene is not only found in mice but also in humans on chromosome 7p11.2–p12. Approximately 10% of patients whom suffer from Russell-Silver Syndrome, a disease associated with severe growth retardation, have a defect in chromosome 7, suggesting a linkage between Grb10 and poor growth in humans.^[11]

In recent news, Grb10 has been dubbed the "hulk protein" due to new findings of a new study from the Garvan Institute of Medical Research in Sydney, Australia. Researchers sought to find the skeletal muscle physiology of mice that had Grb10 deleted from their genome. They found that the enlargement of muscles was due to the increase in numbers of myofibers in the muscle tissue, not myofiber size. ^[12] The investigators also found that the Grb10-deficient mice muscles were metabolically no different then muscles found in wild-type mice. ^[13]

Once these findings were published, the media had a hay day writing about the newly discovered "Hulk" Protein. Here are the titles of a few news articles found online regarding Grb10:

"Scientists Discover 'Hulk' Protein"

"Newly Discovered 'Hulk' Protein Could Make You a Beefcake Without the Weights"

"Death of the gym workouts? 'Hulk' Protein discovered by scientists that could be behind huge muscle growth"

"Growing Strong Muscles Without Working Out? 'Hulk' Protein, Grb10, Controls Muscle Growth"

Dr. Lowenna J. Holt, Ph.D., the lead researcher was quoted saying, "By identifying a novel mechanism regulating muscle development, our work has revealed potential new strategies to increase muscle mass. Ultimately, this might improve treatment of muscle wasting conditions, as well as metabolic disorders such as Type 2 diabetes". Dr. Holt and colleagues did not set out to find a new way to increase the muscle mass for gym rats or competitive athletes but rather a new approach to treat patients that suffer from many different muscle wasting diseases. A lot more research must be done before it can be used on humans, but we may one day see the olympics testing for Grb10 inhibitors for they will most likely be abused as a new modern alternative to steroids.

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4} Stein EG, Ghirlando R, Hubbard SR. Structural basis for dimerization of the Grb10 Src homology 2 domain. Implications for ligand specificity. J Biol Chem. 2003 Apr 11;278(15):13257-64. Epub 2003 Jan 27. PMID:12551896 doi:http://dx.doi.org/10.1074/jbc.M212026200
2. ↑ He W, Rose DW, Olefsky JM, Gustafson TA. Grb10 interacts differentially with the insulin receptor, insulin-like growth factor I receptor, and epidermal growth factor receptor via the Grb10 Src homology 2 (SH2) domain and a second novel domain located between the pleckstrin homology and SH2 domains. J Biol Chem. 1998 Mar 20;273(12):6860-7. PMID:9506989
3. ↑ Huang Q, Szebenyi DM. Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4. J Biol Chem. 2010 Dec 31;285(53):42130-9. Epub 2010 Oct 26. PMID:20980250 doi:10.1074/jbc.M110.143412
4. ↑ Huang Q, Szebenyi DM. Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4. J Biol Chem. 2010 Dec 31;285(53):42130-9. Epub 2010 Oct 26. PMID:20980250 doi:10.1074/jbc.M110.143412
5. ↑ Huang Q, Szebenyi DM. Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4. J Biol Chem. 2010 Dec 31;285(53):42130-9. Epub 2010 Oct 26. PMID:20980250 doi:10.1074/jbc.M110.143412
6. ↑ Huang Q, Szebenyi DM. Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4. J Biol Chem. 2010 Dec 31;285(53):42130-9. Epub 2010 Oct 26. PMID:20980250 doi:10.1074/jbc.M110.143412
7. ↑ Huang Q, Szebenyi DM. Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4. J Biol Chem. 2010 Dec 31;285(53):42130-9. Epub 2010 Oct 26. PMID:20980250 doi:10.1074/jbc.M110.143412
8. ↑ Smith FM, Holt LJ, Garfield AS, Charalambous M, Koumanov F, Perry M, Bazzani R, Sheardown SA, Hegarty BD, Lyons RJ, Cooney GJ, Daly RJ, Ward A. Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life. Mol Cell Biol. 2007 Aug;27(16):5871-86. Epub 2007 Jun 11. PMID:17562854 doi:10.1128/MCB.02087-06
9. ↑ Huang Q, Szebenyi DM. Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4. J Biol Chem. 2010 Dec 31;285(53):42130-9. Epub 2010 Oct 26. PMID:20980250 doi:10.1074/jbc.M110.143412
10. ↑ Morita H, Omichi M, Inami I, Koike S. [Role of hepatic cycle AMP and lysosomal lipase in triglyceridemia induced by injection of cobalt chloride]. Nihon Eiseigaku Zasshi. 1975 Apr;30(1):65. PMID:166222
11. ↑ Smith FM, Holt LJ, Garfield AS, Charalambous M, Koumanov F, Perry M, Bazzani R, Sheardown SA, Hegarty BD, Lyons RJ, Cooney GJ, Daly RJ, Ward A. Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life. Mol Cell Biol. 2007 Aug;27(16):5871-86. Epub 2007 Jun 11. PMID:17562854 doi:10.1128/MCB.02087-06
12. ↑ Holt LJ, Turner N, Mokbel N, Trefely S, Kanzleiter T, Kaplan W, Ormandy CJ, Daly RJ, Cooney GJ. Grb10 regulates the development of fiber number in skeletal muscle. FASEB J. 2012 Sep;26(9):3658-69. doi: 10.1096/fj.11-199349. Epub 2012 May 23. PMID:22623587 doi:10.1096/fj.11-199349
13. ↑ Name= Lowenna PMID: 22623587