Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of ␤-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinintagged AT1aR, angiotensin stimulation triggered ␤-arrestin-2 binding to the receptor and internalization of AT1aR-␤-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-␤-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, ␤-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged ␤-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with ␤-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with ␤-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to ␤-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in ␤-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to ␤-arrestin-2, and the association of ␤-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that ␤-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R ␤ subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR⅐Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu 52 ]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.The insulin receptor family is comprised of three members, the insulin receptor, the insulin-like growth factor-1 receptor (IGF-1R), 1 and the insulin receptor-related receptor, an orphan whose endogenous ligand is unknown. The three receptors share a common topology, each composed of two entirely extracellular ␣ subunits containing the ligand-binding domain, and two ␤ subunits that contain a single transmembrane domain and an intracellular domain possessing intrinsic ligand-stimulated tyrosine kinase activity. Like classical receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), stimulation of insulin receptors leads both to receptor autophosphorylation and to the recruitment and activation of signaling proteins that contain specific phosphotyrosine-binding Src-homology 2 (SH2) domains.In the case of EGFR, phosphorylation of tyrosine residues within the intracellular domain provides docking sites for SH2 domain proteins, including the Ras guanine nucleotide exchange factor complex Grb2⅐mSos, Ras-GTPase-activating protein, phospholipase C␥, the p85⅐p110␣ phosphatidylinositol 3-kinase (PI3K) complex, and the nonreceptor tyrosine kinases c-Src and c-Fyn, as well as for adapter proteins such as Shc and Gab1. Thus the phosphorylated EGFR serves as a scaffold for the assembly, at the plasma membrane, of a multienzymesignaling complex that mediates the intracellular response to EGF (1). Unlike the EGF receptor, autophosphorylated insulin family receptors do not signal via the direct recruitment of these s...

Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin.

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