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Structural and functional studies of the Ras-associating and pleckstrin-homology domains of Grb10 and Grb14.

Depetris RS, Wu J, Hubbard SR - Nat. Struct. Mol. Biol. (2009)

Bottom Line: The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit.Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling.Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, Kimmel Center for Biology and Medicine of the Skirball Institute, and Department of Pharmacology, New York University School of Medicine, New York, New York, USA.

ABSTRACT
Growth factor receptor-binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-A resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

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Crystal structure of human Grb10 RA-PH. (a) Domain architecture of Grb7-10-14 drawn to linear scale (human Grb10, isoform c, 536 residues). The abbreviations are: P, proline-rich; RA, Ras-associating; PH, pleckstrin-homology; BPS, between PH and SH2; and SH2, Src-homology-2. (b) Ribbon diagram of the crystal structure of Grb10 RA-PH. One copy of RA-PH is colored violet (RA) and cyan (PH), and the second copy is colored orange (RA) and green (PH). For both copies, the RA-PH linker is colored gray. In (b) and (c), the binding sites for small GTPases on the RA domain and phosphoinositides on the PH domain (non-canonically) are indicated by the position of the labels ‘RA’ and ‘PH’. An approximate two-fold axis (vertical, in the plane of the figure) relates the two molecules in the asymmetric unit. Select secondary-structure elements are labeled, as are the N- and C-termini. In the right panel, the structure has been rotated 90°, as indicated, with the molecular two-fold axis perpendicular to the plane of the figure. (c) Stereo view of the dimerization interface. The view is the same as in the right panel of (b). Side chains that mediate the interaction between the two RA-PH molecules are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. (d) Stereo view of the interface between the RA and PH domains. Side chains that mediate the interaction between the two domains are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. Figures 1, 3c–d, and 6 were rendered with PyMOL (http://pymol.sourceforge.net).
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Figure 1: Crystal structure of human Grb10 RA-PH. (a) Domain architecture of Grb7-10-14 drawn to linear scale (human Grb10, isoform c, 536 residues). The abbreviations are: P, proline-rich; RA, Ras-associating; PH, pleckstrin-homology; BPS, between PH and SH2; and SH2, Src-homology-2. (b) Ribbon diagram of the crystal structure of Grb10 RA-PH. One copy of RA-PH is colored violet (RA) and cyan (PH), and the second copy is colored orange (RA) and green (PH). For both copies, the RA-PH linker is colored gray. In (b) and (c), the binding sites for small GTPases on the RA domain and phosphoinositides on the PH domain (non-canonically) are indicated by the position of the labels ‘RA’ and ‘PH’. An approximate two-fold axis (vertical, in the plane of the figure) relates the two molecules in the asymmetric unit. Select secondary-structure elements are labeled, as are the N- and C-termini. In the right panel, the structure has been rotated 90°, as indicated, with the molecular two-fold axis perpendicular to the plane of the figure. (c) Stereo view of the dimerization interface. The view is the same as in the right panel of (b). Side chains that mediate the interaction between the two RA-PH molecules are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. (d) Stereo view of the interface between the RA and PH domains. Side chains that mediate the interaction between the two domains are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. Figures 1, 3c–d, and 6 were rendered with PyMOL (http://pymol.sourceforge.net).

Mentions: Grb7, Grb10 and Grb14 are related adapter proteins comprising an N-terminal region containing polyproline stretches, a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 (SH2) domain1,2 (Fig. 1a). Using various biochemical approaches, these adapter proteins have been shown capable of binding to a variety of receptor tyrosine kinases, including the insulin and insulin-like growth factor-1 (IGF1) receptors1,2. Important insights into the true biological roles of Grb10 and Grb14 have come from gene-deletion studies in mice. The Grb10 gene is maternally imprinted in mice, and loss of the maternal allele results in mice that are approximately 30% greater in size than wild-type littermates, with disproportionately large livers3. As adults, these mice exhibit improved glucose tolerance, increased muscle mass and reduced adiposity4,5. Transgenic mice overexpressing Grb10 show postnatal growth retardation and insulin resistance as a consequence of hyper-negative regulation of the insulin and IGF1 receptors6. Male Grb14−/− mice are of normal size and exhibit improved glucose tolerance and enhanced insulin signaling in muscle and liver7. These in vivo studies established that Grb10 and Grb14 are important tissue-specific negative regulators of insulin and IGF1 signaling.


Structural and functional studies of the Ras-associating and pleckstrin-homology domains of Grb10 and Grb14.

Depetris RS, Wu J, Hubbard SR - Nat. Struct. Mol. Biol. (2009)

Crystal structure of human Grb10 RA-PH. (a) Domain architecture of Grb7-10-14 drawn to linear scale (human Grb10, isoform c, 536 residues). The abbreviations are: P, proline-rich; RA, Ras-associating; PH, pleckstrin-homology; BPS, between PH and SH2; and SH2, Src-homology-2. (b) Ribbon diagram of the crystal structure of Grb10 RA-PH. One copy of RA-PH is colored violet (RA) and cyan (PH), and the second copy is colored orange (RA) and green (PH). For both copies, the RA-PH linker is colored gray. In (b) and (c), the binding sites for small GTPases on the RA domain and phosphoinositides on the PH domain (non-canonically) are indicated by the position of the labels ‘RA’ and ‘PH’. An approximate two-fold axis (vertical, in the plane of the figure) relates the two molecules in the asymmetric unit. Select secondary-structure elements are labeled, as are the N- and C-termini. In the right panel, the structure has been rotated 90°, as indicated, with the molecular two-fold axis perpendicular to the plane of the figure. (c) Stereo view of the dimerization interface. The view is the same as in the right panel of (b). Side chains that mediate the interaction between the two RA-PH molecules are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. (d) Stereo view of the interface between the RA and PH domains. Side chains that mediate the interaction between the two domains are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. Figures 1, 3c–d, and 6 were rendered with PyMOL (http://pymol.sourceforge.net).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2748937&req=5

Figure 1: Crystal structure of human Grb10 RA-PH. (a) Domain architecture of Grb7-10-14 drawn to linear scale (human Grb10, isoform c, 536 residues). The abbreviations are: P, proline-rich; RA, Ras-associating; PH, pleckstrin-homology; BPS, between PH and SH2; and SH2, Src-homology-2. (b) Ribbon diagram of the crystal structure of Grb10 RA-PH. One copy of RA-PH is colored violet (RA) and cyan (PH), and the second copy is colored orange (RA) and green (PH). For both copies, the RA-PH linker is colored gray. In (b) and (c), the binding sites for small GTPases on the RA domain and phosphoinositides on the PH domain (non-canonically) are indicated by the position of the labels ‘RA’ and ‘PH’. An approximate two-fold axis (vertical, in the plane of the figure) relates the two molecules in the asymmetric unit. Select secondary-structure elements are labeled, as are the N- and C-termini. In the right panel, the structure has been rotated 90°, as indicated, with the molecular two-fold axis perpendicular to the plane of the figure. (c) Stereo view of the dimerization interface. The view is the same as in the right panel of (b). Side chains that mediate the interaction between the two RA-PH molecules are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. (d) Stereo view of the interface between the RA and PH domains. Side chains that mediate the interaction between the two domains are shown in stick representation. Hydrogen bonds/salt bridges are represented by black dashed lines. The side chains of hydrophobic residues are shown with a van der Waals surface. Figures 1, 3c–d, and 6 were rendered with PyMOL (http://pymol.sourceforge.net).
Mentions: Grb7, Grb10 and Grb14 are related adapter proteins comprising an N-terminal region containing polyproline stretches, a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 (SH2) domain1,2 (Fig. 1a). Using various biochemical approaches, these adapter proteins have been shown capable of binding to a variety of receptor tyrosine kinases, including the insulin and insulin-like growth factor-1 (IGF1) receptors1,2. Important insights into the true biological roles of Grb10 and Grb14 have come from gene-deletion studies in mice. The Grb10 gene is maternally imprinted in mice, and loss of the maternal allele results in mice that are approximately 30% greater in size than wild-type littermates, with disproportionately large livers3. As adults, these mice exhibit improved glucose tolerance, increased muscle mass and reduced adiposity4,5. Transgenic mice overexpressing Grb10 show postnatal growth retardation and insulin resistance as a consequence of hyper-negative regulation of the insulin and IGF1 receptors6. Male Grb14−/− mice are of normal size and exhibit improved glucose tolerance and enhanced insulin signaling in muscle and liver7. These in vivo studies established that Grb10 and Grb14 are important tissue-specific negative regulators of insulin and IGF1 signaling.

Bottom Line: The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit.Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling.Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, Kimmel Center for Biology and Medicine of the Skirball Institute, and Department of Pharmacology, New York University School of Medicine, New York, New York, USA.

ABSTRACT
Growth factor receptor-binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-A resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

Show MeSH