AP-2 adaptors regulate clathrin-bud formation at the cell surface by recruiting clathrin trimers to the plasma membrane and by selecting certain membrane proteins for inclusion within the developing ...clathrin-coat structure. These functions are performed by discrete subunits of the adaptor heterotetramer. The carboxyl-terminal appendage of the AP-2α subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the α appendage at 1.4- angstrom resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a β -sandwich domain and a mixed α -β platform domain. Structure-based mutagenesis shows that alterations to the molecular surface of a highly conserved region on the platform domain differentially affect associations of the appendage with amphiphysin, eps15, epsin, and AP180, revealing a common protein-binding interface.
Rhodopsin, a prototypical G protein-coupled receptor, catalyzes the activation of a heterotrimeric G protein, transducin, to initiate a visual signaling cascade in photoreceptor cells. The βγ subunit ...complex, especially the C-terminal domain of the transducin γ subunit, Gtγ(60–71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin α subunit by light-activated rhodopsin. We report that this domain is unstructured in the presence of an inactive receptor but forms an amphipathic helix upon rhodopsin activation. A K65E/E66K charge reversal mutant of the γ subunit has diminished interactions with the receptor and fails to adopt the helical conformation. The identification of this conformational switch provides a mechanism for active GPCR utilization of the βγ complex in signal transfer to G proteins.
The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gtα and Gtβγ ...subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtγ(60−71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367−373. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin−Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gtγ C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gtγ for alanine. The mutants were co-expressed with six-histidine-tagged Gtβ subunits in Sf9 insect cells. The Gtβ-6-His-γ mutant proteins were purified and assayed in the presence of Gtα for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*−Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gtγ(60−71) region has the most significant effect on rhodopsin−Gt interactions, which places important constraints on the model of the rhodopsin−Gt complex.
Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between ...arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.
Light activated rhodopsin interacts with domains on all three subunits of transducin. Two of these domains, the C-terminal regions of the alpha and gamma subunits mimic the ability of transducin to ...stabilize the active conformation of rhodopsin, metarhodopsin II, but display different roles in transducin activation process. Whether the interactions are with the same or different complimentary sites on Meta II is unknown. We have used chemo-selective thioalkylation of rhodopsin and UV/visible spectroscopy to show that interactions with transducin C-terminal domains can be selectively disrupted. These data provide evidence that formal structural determinants on Meta II for these domains of transducin are different. In a set of complimentary experiments we examined the reactivity of Meta II species produced in the presence of the Gtα and Gtγ subunit peptides to hydroxylamine. Analysis of the rates of Meta II decay confirms that the conformational states of Meta II when bound to Gtα and Gtβγ represent distinct signaling states of rhodopsin.
In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by ...several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein beta subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5' flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.
Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric ...AP-2 adaptor complex by associating directly with the globular appendage of the α subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence 257LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor β subunits, amphiphysin, and β-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the β subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence 480LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.
Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric ...AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.
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