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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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Interaction between Grb7-10-14 and Ras. (a) Co-immunoprecipitation (i.p.) of Myc-tagged Grb7, -10, or -14 with HA-tagged wild-type N-Ras or N-Ras G12D (constitutively activated) from HEK 293T cells. Antibodies used for immunoblotting (i.b.) are indicated on the right sides of the blots, and protein identifications are supplied on the left sides. The bottom blot of the anti-HA immunoprecipitations and the two blots of the lysates are controls for protein levels. (b) Same co-immunoprecipitation experiment as in (a), but with Myc-tagged Grb14, either wild-type or K140A (RA) or K252A (PH), along with wild-type N-Ras or N-Ras G12D.
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Figure 4: Interaction between Grb7-10-14 and Ras. (a) Co-immunoprecipitation (i.p.) of Myc-tagged Grb7, -10, or -14 with HA-tagged wild-type N-Ras or N-Ras G12D (constitutively activated) from HEK 293T cells. Antibodies used for immunoblotting (i.b.) are indicated on the right sides of the blots, and protein identifications are supplied on the left sides. The bottom blot of the anti-HA immunoprecipitations and the two blots of the lysates are controls for protein levels. (b) Same co-immunoprecipitation experiment as in (a), but with Myc-tagged Grb14, either wild-type or K140A (RA) or K252A (PH), along with wild-type N-Ras or N-Ras G12D.

Mentions: Previous pulldown experiments using cell lysates demonstrated that Grb7 (Grb10/14 not tested) binds to activated Ras (N-, K-, and H-Ras) and much weaker to activated Rap1 and Rap226. In a similar fashion, we co-transfected HEK 293T cells with HA-tagged, wild-type N-Ras or a constitutively active mutant (G12D), along with Myc-tagged Grb7, Grb10 or Grb14 (all full-length), immunoprec [0-9]^13 ipitated N-Ras with an anti-HA antibody and immunoblotted for Grb7-10-14 using an anti-Myc antibody. This co-immunoprecipitation experiment (Fig. 4a) demonstrated that Grb14, like Grb7, interacts with N-Ras, but that Grb10 interacts comparatively weakly. This experiment also showed that Grb7-10-14 bind preferentially to activated N-Ras. Pulldown experiments using lysates of HEK 293T cells co-transfected with either constitutively active N-Ras, Rap1 or Rap2 (GST fusions) and Grb10 or Grb14 demonstrated that Grb10 and Grb14 do not bind appreciably to Rap1 or Rap2 relative to N-Ras (data not shown).


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)

Interaction between Grb7-10-14 and Ras. (a) Co-immunoprecipitation (i.p.) of Myc-tagged Grb7, -10, or -14 with HA-tagged wild-type N-Ras or N-Ras G12D (constitutively activated) from HEK 293T cells. Antibodies used for immunoblotting (i.b.) are indicated on the right sides of the blots, and protein identifications are supplied on the left sides. The bottom blot of the anti-HA immunoprecipitations and the two blots of the lysates are controls for protein levels. (b) Same co-immunoprecipitation experiment as in (a), but with Myc-tagged Grb14, either wild-type or K140A (RA) or K252A (PH), along with wild-type N-Ras or N-Ras G12D.
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Figure 4: Interaction between Grb7-10-14 and Ras. (a) Co-immunoprecipitation (i.p.) of Myc-tagged Grb7, -10, or -14 with HA-tagged wild-type N-Ras or N-Ras G12D (constitutively activated) from HEK 293T cells. Antibodies used for immunoblotting (i.b.) are indicated on the right sides of the blots, and protein identifications are supplied on the left sides. The bottom blot of the anti-HA immunoprecipitations and the two blots of the lysates are controls for protein levels. (b) Same co-immunoprecipitation experiment as in (a), but with Myc-tagged Grb14, either wild-type or K140A (RA) or K252A (PH), along with wild-type N-Ras or N-Ras G12D.
Mentions: Previous pulldown experiments using cell lysates demonstrated that Grb7 (Grb10/14 not tested) binds to activated Ras (N-, K-, and H-Ras) and much weaker to activated Rap1 and Rap226. In a similar fashion, we co-transfected HEK 293T cells with HA-tagged, wild-type N-Ras or a constitutively active mutant (G12D), along with Myc-tagged Grb7, Grb10 or Grb14 (all full-length), immunoprec [0-9]^13 ipitated N-Ras with an anti-HA antibody and immunoblotted for Grb7-10-14 using an anti-Myc antibody. This co-immunoprecipitation experiment (Fig. 4a) demonstrated that Grb14, like Grb7, interacts with N-Ras, but that Grb10 interacts comparatively weakly. This experiment also showed that Grb7-10-14 bind preferentially to activated N-Ras. Pulldown experiments using lysates of HEK 293T cells co-transfected with either constitutively active N-Ras, Rap1 or Rap2 (GST fusions) and Grb10 or Grb14 demonstrated that Grb10 and Grb14 do not bind appreciably to Rap1 or Rap2 relative to N-Ras (data not shown).

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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