Abstract
Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including 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) domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V). The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.
Highlights
Grb14 is a member of the Grb7-10-14 family of multi-domain, cytoplasmic adaptor proteins
We determined the crystal structure of a complex between Grb14 RA-PH (Grb14RA-PH) and a constitutively active mutant (G12V) of H-Ras loaded with Mg-GTP
Based on crystal structures and sequence alignments, the integrated RA-PH structural module (Figure 1A) is firmly established in the Grb7-10-14 family of adaptor proteins and in a distinct adaptor-protein family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, which are involved in actin-cyto-skeleton rearrangement [16]
Summary
Grb is a member of the Grb family of multi-domain, cytoplasmic adaptor proteins. Grb is imprinted in mice [8] (and most likely in humans [9]), and loss of the maternal allele results in mice that are approximately 30% greater in overall size than wild-type litter mates, with disproportionately large livers [6]. As adults, these mice exhibit improved glucose tolerance, increased muscle mass, and reduced adiposity [3]. Transgenic mice overexpressing Grb show postnatal growth retardation and insulin resistance as a consequence of hypernegative regulation of the insulin and IGF1 receptors [5]
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