Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis

Background: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs thro ugh its SH2 domain, and engages downstream targets such as Sos1 and Gab1 th rough its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to th e Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3 ' -kinase, By creating a null (Delta) allele of mouse Grb2, we have shown tha t Grb2 is required for endoderm differentiation at embryonic day 4.0. Results: Grb2 likely has multiple embryonic and postnatal functions. To add ress this issue, a hypomorphic mutation, first characterized in the Caenorh abditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gen e. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Fur thermore, animals homozygous for the hypomorphic mutation die perinatally b ecause of clefting of the palate, a branchial arch-derived structure. Analy sis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/M AP kinase activation and Gab1 tyrosine phosphorylation following growth fac tor stimulation. Conclusions: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue m orphogenesis and cell viability necessary for mammalian development. The pl acental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signal ing to the Ras-MAP kinase and Gab1 pathways.