ABSTRACT
Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and ß-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Chimerin Proteins/metabolism , Oncogene Proteins/metabolism , Proto-Oncogene Proteins/metabolism , RNA-Binding Proteins/metabolism , Receptor, EphB2/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Cell Separation , Chimerin Proteins/chemistry , Ephrin-B1/genetics , Ephrin-B1/metabolism , HEK293 Cells , Humans , Mass Spectrometry , Oncogene Proteins/chemistry , Phosphorylation , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA Interference , RNA, Small Interfering/metabolism , RNA-Binding Proteins/chemistry , Receptor, EphB2/chemistry , Receptor, EphB2/genetics , Signal Transduction , src Homology DomainsABSTRACT
Cell signaling depends on dynamic protein-protein interaction (PPI) networks, often assembled through modular domains each interacting with multiple peptide motifs. This complexity raises a conceptual challenge, namely to define whether a particular cellular response requires assembly of the complete PPI network of interest or can be driven by a specific interaction. To address this issue, we designed variants of the Grb2 SH2 domain ("pY-clamps") whose specificity is highly biased toward a single phosphotyrosine (pY) motif among many potential pYXNX Grb2-binding sites. Surprisingly, directing Grb2 predominantly to a single pY site of the Ptpn11/Shp2 phosphatase, but not other sites tested, was sufficient for differentiation of the essential primitive endoderm lineage from embryonic stem cells. Our data suggest that discrete connections within complex PPI networks can underpin regulation of particular biological events. We propose that this directed wiring approach will be of general utility in functionally annotating specific PPIs.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , GRB2 Adaptor Protein/metabolism , Protein Interaction Maps/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Animals , Binding Sites/genetics , Cell Differentiation/genetics , Cell Line , Crystallography, X-Ray , Embryonic Stem Cells/metabolism , Fibroblast Growth Factor 4/metabolism , GRB2 Adaptor Protein/genetics , Mice , Models, Molecular , Protein Binding/genetics , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/ultrastructure , Signal Transduction/geneticsABSTRACT
Metazoan evolution involves increasing protein domain complexity, but how this relates to control of biological decisions remains uncertain. The Ras guanine nucleotide exchange factor (RasGEF) Sos1 and its adaptor Grb2 are multidomain proteins that couple fibroblast growth factor (FGF) signaling to activation of the Ras-Erk pathway during mammalian development and drive embryonic stem cells toward the primitive endoderm (PrE) lineage. We show that the ability of Sos1/Grb2 to appropriately regulate pluripotency and differentiation factors and to initiate PrE development requires collective binding of multiple Sos1/Grb2 domains to their protein and phospholipid ligands. This provides a cooperative system that only allows lineage commitment when all ligand-binding domains are occupied. Furthermore, our results indicate that the interaction domains of Sos1 and Grb2 have evolved so as to bind ligands not with maximal strength but with specificities and affinities that maintain cooperativity. This optimized system ensures that PrE lineage commitment occurs in a timely and selective manner during embryogenesis.
Subject(s)
Embryo, Mammalian/metabolism , Embryonic Stem Cells/metabolism , GRB2 Adaptor Protein/metabolism , SOS1 Protein/metabolism , Amino Acid Sequence , Animals , Cell Lineage , Endoderm/metabolism , Eukaryota/genetics , Eukaryota/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment , ras Guanine Nucleotide Exchange Factors/metabolismABSTRACT
Growth factor-induced receptor dimerization and cross-phosphorylation are hallmarks of signal transduction via receptor tyrosine kinases (RTKs). G protein-coupled receptors (GPCRs) can activate RTKs through a process known as transactivation. The prototypical model of RTK transactivation involves ligand-mediated RTK dimerization and cross-phosphorylation. Here, we show that the platelet-derived growth factor receptor beta (PDGFRbeta) transactivation by the dopamine receptor D4 (DRD4) is not dependent on ligands for PDGFRbeta. Furthermore, when PDGFRbeta dimerization is inhibited and receptor phosphorylation is suppressed to near basal levels, the receptor maintains its ability to be transactivated and is still effective in signaling to ERK1/2. Hence, the DRD4-PDGFRbeta-ERK1/2 pathway can occur independently of a PDGF-like ligand, PDGFRbeta cross-phosphorylation and dimerization, which is distinct from other known forms of transactivation of RTKs by GPCRs.
Subject(s)
Protein Multimerization , Receptor, Platelet-Derived Growth Factor beta/genetics , Receptor, Platelet-Derived Growth Factor beta/metabolism , Receptors, Dopamine D4/metabolism , Transcriptional Activation/genetics , Animals , Becaplermin , Cell Line , Dopamine/pharmacology , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mice , Paracrine Communication/drug effects , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Platelet-Derived Growth Factor/pharmacology , Protein Multimerization/drug effects , Proto-Oncogene Proteins c-sis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Transcriptional Activation/drug effectsABSTRACT
Dopamine receptors are GPCRs that play important roles in locomotion, reward, and cognitive processes. Previously, we demonstrated that this receptor transactivates PDGFRbeta to modulate ERK1/2 and NMDA receptor activity. Downregulation of maturely glycosylated PDGFRbeta by prolonged exposure to PDGF-BB eliminated PDGF-BB-mediated ERK1/2 activation. The DRD4-mediated ERK1/2 response was only partially blunted by PDGF-BB-mediated downregulation, but remained sensitive to the PDGFRbeta kinase inhibitor tyrphostin A9. Tunicamycin prevented the N-linked glycosylation and maturation of PDGFRbeta as well as its activation by PDGF-BB. However, upon tunicamycin treatment, DRD4 continued to signal to ERK1/2 in a tyrphostin A9-sensitive manner. Collectively, our observations indicate that DRD4, unlike PDGF-BB, can activate a pool of intracellularly located PDGFRbeta.