Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

We have examined the endocytic trafficking of epitopetagged ␦ and opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1 ⁄2 ϳ 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either ␦ or opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.Opioid receptors constitute a class of G protein-coupled receptors that mediate the effects of endogenously produced opioid peptides in the central and peripheral nervous systems. An interesting feature of these receptors is that they are activated both by native peptides and by structurally distinct non-peptide alkaloid ligands (1). Following activation, opioid receptors are regulated by multiple mechanisms, which modulate the functional plasticity of the endogenous opioid system and contribute to the development of opiate tolerance and dependence (2-5).Previous studies have described two distinguishable processes of opioid receptor regulation, termed desensitization and down-regulation (3, 4, 6 -9). Both desensitization and downregulation of opioid receptors are stimulated by peptide and alkaloid agonists (4, 6). However, individual agonists may have substantially different effects on the rapid regulation of opioid receptors (10 -12). We have observed that opioid receptors are regulated by a process of rapid internalization that exhibits a remarkable degree of agonist specificity not observed previously in studies of the intracellular trafficking of other receptors. EXPERIMENTAL PROCEDURES Construction and Expression of Epitope-tagged ␦ and OpioidReceptors-cDNAs encoding murine ␦ (13) and (14) opioid receptors were epitope-tagged in the amino-terminal extracellular domain utilizing a shuttle vector containing a signal-FLAG cassette (kindly provided by Drs. Jeff Reagan and Brian Kobilka) (15) and subcloned into pcDNA3 (Invitrogen) for transfection. The structure of each mutant cDNA was confirmed by dideoxy sequencing (Sequenase, U. S. Biochemical Corp.).Human embryonic kidney (HEK) 1 293 cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (University of California San Francisco Cell Culture Facility) and transfected using calcium phosphate coprecipitation (16). Stably transfected cells were isolated following neomycin selection (Geneticin, Life Technologies, Inc.) as described previously (17), and satur...

A composite time series (1993–2000) of sea surface height anomaly from several satellites is used to identify eddies in the South China Sea (SCS). The eddy lifetime, radius, strength, and straight‐line travel distance are estimated. Altogether 58 anticyclonic eddies and 28 cyclonic eddies are identified for this period. They are grouped into four geographical zones according to known eddy generation mechanisms, and their statistics are discussed accordingly. Our geographical classification is a useful first step in gaining an overview of their generation.

mu-Opioid receptors are the pharmacological targets of endogenous opioid peptides and morphine-like alkaloid drugs. Previous studies of transfected cells and peripheral neurons indicate that opioid receptors are rapidly internalized after activation by the alkaloid agonist etorphine but not after activation by morphine. To determine whether opioid receptors in the central nervous system are regulated by a similar process of agonist-selective internalization, mu-opioid receptors were examined in rat brain neurons after treatment of animals with opioid drugs. Internalized mu receptors were observed within 30 min after intraperitoneal injection of the alkaloid agonist etorphine, and this process was blocked by the antagonist naloxone. Colocalization of internalized opioid receptors with transferrin receptors in confocal optical sections indicated that receptor internalization observed in vivo is mediated by a membrane trafficking pathway similar to that observed previously in vitro using transfected human embryonic kidney 293 cells. Morphine failed to induce detectable rapid internalization of receptors, even when administered to animals at doses far in excess of those required to induce analgesia. To quantify these agonist-selective differences and to analyze an array of opioid ligands for their ability to trigger internalization, we used flow cytometry on stably transfected 293 cells. These studies indicated that the different effects of individual agonists are not correlated with their potencies for receptor activation and that a variety of clinically important agonists differ significantly in their relative abilities to stimulate the rapid internalization of opioid receptors.