The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, /-and PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aPKCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.

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Front cover (paperback): South wall of a mural depicting Detroit Industry, 1932-33 (fresco), Rivera, Diego (1886-1957)/Detroit Institute of Arts, USA/Gift of Edsel B. Ford/Bridgeman Images. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Cold Spring Harbor xiv Foreword The year 2015 marked the sesquicentennial anniversary of Gregor Mendel's landmark 1865 presentation of his paper "Experiments on Plant Hybridization," which laid the groundwork for modern genetics. Seminal discoveries throughout the 20th century followed, not least of which was the demonstration that chromatin carried traits, the formalization of the concept of the gene as the hereditary unit, the discovery of DNA as the hereditary material, the discovery of the double helix structure of DNA, and the elucidation of many mechanisms now known to operate to express and protect the hereditary material in its nuclear, cellular, and organismal context. The year also marked the 125th anniversary of Cold Spring Harbor Laboratory as well as the 80th Cold Spring Harbor Laboratory Symposium on Quantitative Biology, the preeminent and storied series of landmark meetings initiated by Reginald Harris in 1933. It therefore seemed fitting to focus this year's Symposium on 21st Century Genetics: Genes at Work to provide a current synthesis of genetic mechanisms and genome/chromosome biology. The decision to plan the 2015 Symposium on this theme reflects the enormous research progress achieved in recent years, and it was intended to provide a broad synthesis of the current state of the field, setting the stage for future discoveries and application. Opening night speakers included Robert Tjian (HHMI and UC Berkeley) on probing transcription regulation by single-molecule imaging; Denis Duboule (EPF Lausanne, Switzerland), who spoke about long-range regulation during development and evolution; Ron Evans (Salk Institute for Biological Studies) on nuclear receptors-feast, famine, and physiology; and Angelika Amon (Massachusetts Institute of Technology), who addressed the effects of aneuploidy on cellular fitness and tumorigenesis. Svante Pääbo (Max Planck Institute for Evolutionary Anthropology, Germany) addressed The Genetic Legacy of Neanderthals in an outstanding Dorcas Cummings Lecture for Laboratory friends, neighbors, and Symposium participants in advance of the annual dinner parties. Susan Gasser provided a masterful Summary at the conclusion of the Symposium immediately before the final banquet. This Symposium was attended by almost 350 scientists from universities around the country and the world, and the program included 65 invited presentations and 135 poster presentations. To disseminate the latest results and discussion of the Symposium to a wider audience, attendees were able to share many of the Symposium talks with their colleagues who were unable to attend using the Leading Strand video archive.

5-Hydroxytryptamine 2A (5-HT2A) receptors are essential for the actions of serotonin (5-hydroxytryptamine (5-HT)) on physiological processes as diverse as vascular smooth muscle contraction, platelet aggregation, perception, and emotion. In this study, we investigated the molecular mechanism(s) by which 5-HT activates 5-HT2A receptors using a combination of approaches including site-directed mutagenesis, molecular modeling, and pharmacological analysis using the sensitive, cell-based functional assay R-SAT. Alaninescanning mutagenesis of residues close to the intracellular end of H6 of the 5-HT2A receptor implicated glutamate Glu-318(6.30) in receptor activation, as also predicted by a newly constructed molecular model of the 5-HT2A receptor, which was based on the x-ray structure of bovine rhodopsin. Close examination of the molecular model suggested that Glu-318(6.30) could form a strong ionic interaction with Arg-173(3.50) of the highly conserved "(D/E)RY motif" located at the interface between the third transmembrane segment and the second intracellular loop (i2). A direct prediction of this hypothesis, that disrupting this ionic interaction by an E318(6.30)R mutation would lead to a highly constitutively active receptor with enhanced affinity for agonist, was confirmed using R-SAT. Taken together, these results predict that the disruption of a strong ionic interaction between transmembrane helices 3 and 6 1 receptors are essential for the actions of serotonin (5-hydroxytryptamine; 5-HT) on a number of key physiological processes including platelet aggregation, vascular and nonvascular smooth muscle contraction, perception, and emotion (1). Additionally, 5-HT2A receptors represent a major site of action of hallucinogens such as lysergic acid diethylamide, which are agonists, and atypical antipsychotic drugs such as clozapine, which are antagonists (2). 5-HT2A receptors are unique in that both agonists and antagonists induce receptor internalization (3) that is dynamin-dependent and arrestin-independent (4). Despite considerable study (5-9), the molecular and atomic mechanisms by which 5-HT induces activation of the 5-HT2A receptor or of the other 15 cloned 5-HT receptors are currently unknown.Prior studies have suggested several potential models of agonist-induced activation of 5-HT receptors in particular and other G-protein-coupled receptors (GPCRs) in general. Initial site-directed mutagenesis studies of the 5-HT2A (10) and ␣ 1b -adrenergic receptors (11, 12) as well as rhodopsin (13) implicated a negatively charged residue in transmembrane helix 3 (H3), which could form a strong interaction with positively charged/polar residues in H7 ("H3-H7 interaction model"). Another model ("H2-H7 proximity model") (7) suggested that hydrogen bonding interactions between H2 and H7 were most important for the activation of 5-HT2A receptors and gonadotrophic hormone receptors (14). Predictions based on the H2-H7 proximity model suggest that this could be a general model for GPCR activation (14,15). More recent studies...

Site-directed mutagenesis and molecular modeling were used to investigate the molecular interactions involved in ligand binding to, and activation of, the rat 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin (5-HT) receptor. Based on previous modeling studies utilizing molecular mechanics energy calculations and molecular dynamics simulations, four sites (S239[5.43], F240[5.44], F243[5.47], and F244[5.48]) in transmembrane region V were selected, each predicted to contribute to agonist and/or antagonist binding. The F243A mutation increased the affinity of (+/-)4-iodo-2, 5-dimethoxyphenylisopropylamine, decreased the binding of alpha-methyl-5HT, N-omega-methyl-5HT, ketanserin, ritanserin, and spiperone and had no effect on the binding of 5-HT and 5-methyl-N, N-dimethyltryptamine. The F240A mutant had no effect on the binding of any of the ligands tested, whereas F244A caused an agonist-specific decrease in binding affinity (3- to 10-fold). S239A caused a 6- to 13-fold decrease in tryptamine-binding affinity and a 5-fold increase in affinity of 4-iodo-2, 5-dimethoxyphenylisopropylamine. A subset of the agonists used in binding studies were used to determine the efficacies and potencies of these mutants to activate phosphoinositide hydrolysis. The F243A and F244A mutations reduced agonist stimulated phosphoinositide hydrolysis, whereas the S239A and F240A mutations had no effect. There was little correlation between agonist binding and second messenger production. Furthermore, molecular dynamics simulations, considering these data, produced ligand-bound structures utilizing substantially different bonding interactions even among structurally similar ligands (differing by as little as one methyl group). Taken together, these results suggest that relatively minor changes in either receptor or ligand structure can produce drastic and unpredictable changes in both binding interactions and 5-HT(2A) receptor activation. Thus, our finding may have major implications for the future and feasibility of receptor structure-based drug design.