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-2 ␣ subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the ␣ appendage at 1.4-Å resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a ␤-sandwich domain and a mixed ␣-␤ 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.Eukaryotic cells take up extracellular macromolecules within small invaginations of the cell surface in a process termed endocytosis. The bulk of endocytosis is believed to occur at discrete bud sites on the plasma membrane coated with a polygonal clathrin lattice. AP-2 adaptors play a pivotal role in the assembly of these clathrin-coated buds (1, 2). The heterotetrameric adaptor, composed of Ϸ100-kDa ␣-and ␤2-, 50-kDa 2-and 17-kDa 2 subunits (Fig. 1A), recruits clathrin trimers to the membrane surface, orchestrating the assembly of the clathrin lattice. AP-2 also plays a direct role in selecting molecules for preferential inclusion within the developing clathrin-coated bud (1). The sorting function appears to be mediated primarily by the 2 subunit of the adaptor complex. 2 interacts directly, albeit weakly, with tyrosine-based internalization signals present in many transmembrane proteins (3). The 2 subunit has also been reported to interact directly with the dileucine class of sorting signals (4). The crystal structures of the carboxyl-terminal portion of the 2 chain associated with peptides containing tyrosinebased internalization signals show that these sorting signals interact with 2 in an extended conformation (5). Because AP-2 maintains direct interactions with both the overlying clathrin lattice and the sorting signals projecting up from the membrane below, the adaptor is incorporated into clathrin-coated vesicles near stoichiometrically with clathrin.It is now clear that additional proteins also contribute to the productive assembly of clathrin-coated vesicles. For example, the GTP-binding protein dynamin plays an important role in the final scission process (6). Dynamin appears to be recruited into the assembling lattice by amphiphysin; a proline-based sequence (PSRPNR), located at the carboxyl-terminal end of dynamin, binds to the Src homology 3 (SH3) domain of amphiphysin. The crystal structure of the amphiphysin II SH3 domain reveals that good specificity is achieved for these two binding partners by alteration of the general SH3 fold in amphiphysin to ...

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 ␣ 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 LMD-LADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor ␤ subunits, amphiphysin, and ␤-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 ␤ 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.Endocytosis occurs primarily at specific regions of the plasma membrane coated on the cytoplasmic surface with the AP-2 adaptor complex and clathrin. The ordered polymerization of clathrin into a polyhedral coat is thought to mechanically introduce curvature into the underlying membrane and thereby drive the formation of clathrin-coated vesicles. Because AP-2 and clathrin are the major protein components on clathrin-coated vesicles that bud from the cell surface, much work over the past decade has centered on carefully dissecting the specific roles that these proteins play in ordered coat assembly and the protein sorting process. It has become clear, however, that multiple factors in addition to clathrin and AP-2 are critical in regulating and coordinating endocytic events. Some additional molecules that have been demonstrated to affect endocytosis include dynamin (1, 2), amphiphysin (3, 4), eps15 (5-7), ␤-arrestin (8), epsin (9), intersectin/Ese (10, 11), synaptojanin (12), POB1 (13), and polyphosphoinositides (14 -18).Although dynamin and synaptojanin each exhibit hydrolytic (GTPase and inositol-5-phosphatase, respectively) activity, the other endocytic proteins appear to be primarily involved in establishing an extensive array of complex ...

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 betagamma subunit complex, especially the C-terminal domain of the transducin gamma subunit, Gtgamma(60-71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin alpha 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 gamma 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 betagamma complex in signal transfer to G proteins.

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 helixloop 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.Following activation by a variety of sensory stimuli, such as hormones, neurotransmitters, or light, G-protein-coupled receptors (GPCRs) 1 are deactivated by multiple phosphorylations and subsequent binding of a regulatory protein arrestin (1, 2). Deactivation of the active receptor is obligatory and ensures the quantum character of the response to an extracellular signal. GPCR kinases (GRKs) and arrestin proteins are receptor-specific but share universal mechanisms of action, with prototypical rhodopsin kinase and visual arrestin involved in the termination of visual signal transduction (3, 4). Quenching of the photoresponse in the retinal photoreceptor cells proceeds through phosphorylation of multiple serine and threonine residues at the C terminus of light-activated rhodopsin by GRK1 and high affinity binding of visual arrestin. In vitro studies have implicated all seven serine and threonine residues within the Rh-(334 -343) C-terminal stretch as possible substrates of the phosphorylation reaction. The question of the exact number and position of residues phosphorylated in vivo, however, remains uncertain possibly due to the inherent difficulties of controlling dephosphorylation, different kinetics of phosphorylation/dephosphorylation at individual residues, possible contribution of kinases other than GRK1, and other secondary factors. Either Ser-334 (5) or Ser-343 (6) is phosphorylated initially. The majority of studies provides strong evidence, however, that multiple phosphorylation is required for reproducible deactivation (5-8). In order to circumvent the current uncertainty about the exact sequence of phosphorylation events, we have used a model synthetic peptide phosphorylated at all sev...

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 Gtalpha and Gtbetagamma subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtgamma(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 Gtgamma 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 Gtgamma for alanine. The mutants were co-expressed with six-histidine-tagged Gtbeta subunits in Sf9 insect cells. The Gtbeta-6-His-gamma mutant proteins were purified and assayed in the presence of Gtalpha 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 Gtgamma(60-71) region has the most significant effect on rhodopsin-Gt interactions, which places important constraints on the model of the rhodopsin-Gt complex.