Barbara A. Lyons

Alternative splicing of the voltage-gated Ca2+ channel β4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex

Ca2+ channel β subunits regulate cell-surface expression and gating of voltage-dependent Ca2+ channel α1 subunits. Based on primary sequence comparisons, β subunits are predicted to be modular structures composed of five domains (A–E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the β subunit core B–D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca2+ channel β subunits (β1–β4) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved β4a A domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Cav2.1 Ca2+channel gating but serves as a binding site in protein–protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to β4 subunits, the interactions are specific for the β4a splice variant, because they do not occur with the β4b A domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca2+ channel β subunits and the cell-specific roles they play in the CNS.

The cell migration protein Grb7 associates with transcriptional regulator FHL2 in a Grb7 phosphorylation-dependent manner

Grb7 is an adaptor molecule that can mediate signal transduction from multiple cell surface receptors to various downstream signaling pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase (RTK), erbB2, are overexpressed in 20–30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, though the participants in these pathways have not been determined. In this study, we report that Grb7 interacts with four and half lim domains isoform 2 (FHL2), a transcription regulator with an important role in oncogenesis, including breast cancer. Additionally, in yeast 2‐hybrid (Y2H) assays, we show that the interaction is specific to the Grb7 RA and PH domains. We have also demonstrated that full‐length (FL) Grb7 and FHL2 interact in mammalian cells and that Grb7 must be tyrosine phosphorylated for this interaction to occur. Immunofluorescent microscopy demonstrates possible co‐localization of Grb7 and FHL2. A model with supporting NMR evidence of Grb7 autoinhibition is proposed. Copyright

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel β4a subunit

Ca2+ channel β subunits regulate trafficking and gating (opening and closing) of voltage‐dependent Ca2+ channel α 1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A–E) that are related to the large family of membrane associated guanylate‐kinase (MAGUK) proteins. The crystal structures of the β subunit core, B–D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N‐terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel β subunits (β1–β4). Furthermore, this domain undergoes alternative splicing to create multiple N‐terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human β4a subunit, a splice variant that we have shown previously to have α 1 subunit subtype‐specific effects on Ca2+ channel trafficking and gating.

Backbone nuclear relaxation characteristics and calorimetric investigation of the human Grb7-SH2/erbB2 peptide complex.

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20%–30% of breast cancers. Grb7 binds to erbB2 and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In a prior study, we reported the solution structure of the Grb7‐SH2/erbB2 peptide complex. In this study, T1, T2, and steady‐state NOE measurements were performed on the Grb7‐SH2 domain, and the backbone relaxation behavior of the domain is discussed with respect to the potential function of an insert region present in all three members of this protein family. Isothermal titration calorimetry (ITC) studies were completed measuring the thermodynamic parameters of the binding of a 10‐residue phosphorylated peptide representative of erbB2 to the SH2 domain. These measurements are compared to calorimetric studies performed on other SH2 domain/phosphorylated peptide complexes available in the literature.

Grb7 binds to Hax-1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation

Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7‐mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been fully determined. In this study, we report that Grb7 interacts with Hax‐1, a cytoskeletal‐associated protein found overexpressed in metastatic tumors and cancer cell lines. Additionally, in yeast 2‐hybrid assays, we show that the interaction is specific to the Grb7‐RA and ‐PH domains. We have also demonstrated that full‐length Grb7 and Hax‐1 interact in mammalian cells and that Grb7 is tyrosine phosphorylated. Isothermal titration calorimetry measurements demonstrate the Grb7‐RA‐PH domains bind to the Grb7‐SH2 domain with micromolar affinity, suggesting full‐length Grb7 can exist in a head‐to‐tail conformational state that could serve a self‐regulatory function. Copyright

Dimerization in the Grb7 protein.

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor–bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin‐interacting protein, human HS‐1‐associated protein‐1. Here, we present results describing the importance of dimerization in the Grb7–Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation‐mimic mutant (Y80E–Grb7–SH2) is largely dimerization deficient and binds a tyrosine‐phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild‐type SH2 domain. Another dimerization‐deficient mutant (F99R–Grb7–SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E–Grb7–SH2 and F99R–Grb7–SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type–Grb7–SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7–SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Copyright

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

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase, ErbB2, are overexpressed in 20–30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin‐a, an actin‐crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA‐PH domains of Grb7, and the immunoglobulin‐like repeat 16–19 domains of Flna. We demonstrate that full‐length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co‐localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling. Copyright

Grb7 protein RA domain oligomerization

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl‐1, Mec‐3 (LIM) domains isoform 2 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 ras associating and pleckstrin homology 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, nuclear magnetic resonance, 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.

Grb7 and Hax1 may colocalize partially to mitochondria in EGF‐treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1

Growth factor receptor bound protein 7 (Grb7) is a signal‐transducing adaptor protein that mediates specific protein–protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein‐binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras‐GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti‐apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor‐treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time‐dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright

Water-Refined Solution Structure of the Human Grb7-SH2 Domain in Complex with the erbB2 Receptor Peptide pY1139

We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J. Mol. Graph., 1990, 8(1), 52-56) falling closer to expected values for well-refined structures.