Grb7 and Filamin‐a associate and are colocalized to cell membrane ruffles upon EGF stimulation

Paudyal, Prakash; Shrestha, Sanjay; Madanayake, Thushara W.; Shuster, Charles B.; Rohrschneider, Larry R.; Rowland, Aaron M; Lyons, Barbara

doi:10.1002/jmr.2297

Cited by 14 publications

(11 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This could occur without outright disengagement of full domains from the overall globular structure. Future work focused on measuring the binding characteristics of Grb7 phosphorylation mimics with known downstream signaling partners such as Filamin‐a may prove enlightening in understanding this process.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

Bradford¹,

Koirala

Park

et al. 2019

J of Molecular Recognition

Self Cite

View full text Add to dashboard Cite

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerizationdependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways. Extrapolation to the full-length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosinephosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated-tyrosine-mimic Y492E-FL-Grb7 protein (Y492E-FL-Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild-type FL Grb7(WT-FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization K d of WT-FL-Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT-FL-Grb7 in comparison the tyrosine-phosphorylation mimic Y492E-FL-Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation. Abbreviations: 6His, six histidine residue protein tag used for purification; AUC, analytical ultracentrifugation; BPS, between pleckstrin and SH2; DLS, dynamic light scattering; DTT, dithiothreitol;ErbB2, erythroblastosis oncogene B2; FL, full length; Grb7, growth factor receptor-bound protein 7; GST, glutathione S-transferase protein tag used for purification; MES, 2-(N-morpholino) ethanesulfonic acid; PH, pleckstrin homology; RA, Ras associating; SEC, size exclusion chromatography; SH2, Src homology 2; Ve/Vo, the ratio of the elution volume to the void volume of the column; WT, wild type

show abstract

Section: Discussionmentioning

confidence: 99%

“…The RA‐PH domain region is important in protein‐protein interactions and can act as a discreet functional unit . There is evidence this region is also capable of oligomerization.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

Bradford¹,

Koirala

Park

et al. 2019

J of Molecular Recognition

Self Cite

View full text Add to dashboard Cite

show abstract

“…Studies in mice show that FLNa stabilises actin filaments in fibroblasts and mediates wound closure by promoting elastic deformation and maintenance of tension in the collagen matrix [53]. FLNa accumulates at membrane ruffles where it interacts with different binding proteins and regulates fibroblast interactions with their mechanical environment [54]. When FLNa was blocked using short hairpin RNA, fibroblasts were unable to maintain tension in collagen matrices, and they had reduced migration in vitro.…”

Section: Filamin-amentioning

confidence: 99%

The Role of Actin Remodelling Proteins in Wound Healing and Tissue Regeneration

Kopecki¹,

Cowin²

2016

Wound Healing - New Insights Into Ancient Challenges

View full text Add to dashboard Cite

The actin cytoskeleton is an essential network of ilaments that is found in all cells and has an important role in regulating cellular activities. The dynamic regulation of cytoskeletal synthesis, remodelling and function is critical for many physiological processes and is integral for the successful repair of wounds. Wound healing relies on the ine balance between cellular proliferation, adhesion and migration, resulting in tightly controlled equilibrium between tissue regeneration and ibrosis. The actin cytoskeleton regulates all these processes and is therefore an important factor contributing to the re-establishment of the skin barrier function, restoration of the skin anatomical structure and wound repair however, it also inevitably results in scar formation. Regulation of the actin cytoskeleton is tightly controlled by several large protein families, which are discussed in this chapter. Members of the FERM superfamily of proteins, the ilamin and tropomyosin families of actin-associated proteins as well as the gelsolin family of actin remodelling proteins are all important regulators of the actin cytoskeleton, which can afect diferent stages of wound healing. Targeted therapies against diferent proteins involved in cytoskeletal regulation may lead to novel therapeutic interventions aimed at improving wound healing and reducing scar formation.

show abstract

“…The RAPH domain region is important in protein‐protein interactions and can act as a discreet functional unit . There is evidence this region is also capable of oligomerization.…”

Section: Introductionmentioning

confidence: 99%

Grb7 protein RA domain oligomerization

Godamudunage

Foster

Warren

et al. 2017

J of Molecular Recognition

Self Cite

View full text Add to dashboard Cite

The Grb7 protein is an adaptor protein that is often co-amplified with the ErbB2 receptor in 20–30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The RA and PH domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half LIM domains isoform 2 (FHL2) and filamin α. These interactions are believed to play a role in regulating Grb7 mediated cell migration function. The full-length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the RA and PH domains, and the importance of this oligomerization in Grb7 function is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, NMR nuclear relaxation studies, glutaraldehyde cross linking and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms, and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.

show abstract

Grb7 and Filamin‐a associate and are colocalized to cell membrane ruffles upon EGF stimulation

Cited by 14 publications

References 29 publications

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

The Role of Actin Remodelling Proteins in Wound Healing and Tissue Regeneration

Grb7 protein RA domain oligomerization

Contact Info

Product

Resources

About