Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites.

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.

Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly.

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 alpha subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the alpha appendage at 1.4-A resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a beta-sandwich domain and a mixed alpha-beta 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.

Mot3, a Zn finger transcription factor that modulates gene expression and attenuates mating pheromone signaling in Saccharomyces cerevisiae.

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.

Community patterns of psychiatric disorders after the Exxon Valdez oil spill.

OBJECTIVE: This study examined the relationship between exposure to the Exxon Valdez oil spill and subsequent cleanup efforts and the prevalence of generalized anxiety disorder, posttraumatic stress disorder (PTSD), and depressive symptoms in 13 Alaska communities. METHOD: A community survey of 599 men and women was conducted approximately 1 year after the spill occurred. Questions from the National Institute of Mental Health Diagnostic Interview Schedule were used to assess symptoms of generalized anxiety disorder and PTSD. The Center for Epidemiologic Studies Depression (CES-D) Scale was used to assess levels of depressive symptoms. RESULTS: The post-spill (i.e., 1-year) prevalence of generalized anxiety disorder and PTSD for the study communities with all degrees of exposure was 20.2% and 9.4%, respectively. The prevalence of respondents with CES-D Scale scores above 16 and 18 was 16.6% and 14.2%, respectively. When compared with the unexposed group, members of the high-exposure group were 3.6 times as likely to have generalized anxiety disorder, 2.9 times as likely to have PTSD, 1.8 times as likely to have a CES-D Scale score of 16 and above, and 2.1 times as likely to have a CES-D Scale score of 18 and above. Women exposed to this event were particularly vulnerable to these conditions, and Alaska Natives were particularly vulnerable to depressive symptoms after the oil spill. CONCLUSIONS: The results suggest that the oil spill's impact on the psychosocial environment was as significant as its impact on the physical environment. The Exxon Valdez experience suggests a number of implications for the mental health needs of disaster victims, particularly in primary care settings.

Ethnic differences in stress, coping, and depressive symptoms after the Exxon Valdez oil spill.

This study assessed levels of depressive symptomatology in a household probability sample of Alaskan Native (N = 188) and Euro-American (N = 371) residents of 13 communities in Alaska. Our objective was to examine ethnic differences in both the association between depressive symptomatology and exposure to the Exxon Valdez oil spill and subsequent cleanup efforts, and in the role of family support as a moderator of exposure to this technological disaster. Level of exposure was significantly associated with mean Center for Epidemiological Studies-Depression Scale scores in both Natives (p less than .05) and Euro-Americans (p less than .01). Both ethnic groups also reported significant declines in traditional relations with increasing levels of exposure (p less than .001). However, Natives had a significantly higher mean Exposure Index score than Euro-Americans and were more likely to report working on cleanup activities, damage to commercial fisheries, and effects of the spill on subsistence activities. Depressive symptomatology was associated with reported participation in cleanup activities and other forms of contact with the oil in Natives, and reported damage to commercial fisheries, use of affected areas, and residence in a community in geographic proximity to the spill in Euro-Americans. Perceived family support was not directly associated with depressive symptoms in either ethnic group, but did serve to buffer the effects of exposure on depressive symptoms in Euro-Americans. The results suggest that cultural differences play an important role in determining the psychosocial impacts of a technological disaster, particularly with respect to exposure, appraisal of an event as stressful, perceived family support as a moderator of stress, and expression of depressive symptomatology.

Interferon-induced reversible acute renal failure with nephrotic syndrome.

Although there has been considerable experience with interferons in clinical trials during the past decade, acute renal failure as a side effect of interferon treatments has rarely been reported. We report a case in which acute renal failure with nephrotic syndrome was associated with therapy with two types of interferons. We note incomplete return of renal function upon withdrawal of therapy.