CSM News Electronic Edition Volume 7, number 11 October 19, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Random Mutagenesis of the Chemoattractant Receptor, cAR1, of Dictyostelium: Evidence for Multiple States of Activation. Jacqueline L. S. Milne, Michael J. Caterina and Peter N. Devreotes Department of Biological Chemistry, The Johns Hopkins School of Medicine Baltimore, MD 21205 J. Biol. Chem., in press. SUMMARY cAMP receptor 1 (cAR1) of Dictyostelium couples to the G protein G2 to mediate activation of adenylyl and guanylyl cyclases, chemotaxis and cell aggregation. Other cAR1-dependent events, including receptor phosphorylation and influx of extracellular Ca2 +, do not require G proteins. To further characterize signal transduction through cAR1, we performed random mutagenesis of the third intracellular loop (twenty-four amino acids) since the corresponding region of other seven helix receptors has been impli cated in the coupling to G proteins. Mutant receptors were expressed in car1- cells and were characterized for G protein-dependent and -independent signal transduction. Our results demonstrate that cAR1 is remarkably tolerant to amino acid substitutions in the third intracellular loop. Of the twenty-one positions where amino acid substitutions were observed, one or more replacements were found that retained full biological function. However, certain alterations resulted in receptors with reduced abili ty to bind cAMP and/or transduce signals. There were specific signal transduction mutants that could undergo cAMP-dependent cAR1 phosphorylation but were impaired either in coupling to G proteins, in G protein-independent Ca2+ influx, or in both pathways . In addition, there were general activation mutants that failed to restore aggregation to car1- cells and displayed severe defects in all signal transduction events, including the most robust response, cAMP-dependent cAR1 phosphorylation. Certain of these mutant phenotypes were obtained in a complementary study, where the entire region of cAR1 from the third to the seventh transmembrane helices was randomly mutagenized. Considered together, these studies indicate that the activation cycle of cAR1 may involve a number of distinct receptor intermediates. A model of G protein-dependent and -independent signal transduction through cAR1 is discussed. -------------------------------------------------------------------- Random Mutagenesis of the Chemoattractant Receptor, cAR1 of Dictyostelium: Mutant Classes that Cause Discrete Shifts in Agonist Affinity and Lock the Receptor in a Novel Activational Intermediate Kim, J.Y1, Caterina, M.J.1, 2, Milne, J.L.S., Lin, K.C., Borleis, J.A., and Devreotes, P.N.3 Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, MD 21205 J. Biol. Chem., in press Summary, The chemoattractant receptor, cAR1, of Dictyostelium transduces extracellular cAMP signals via G-protein dependent and G-protein independent mechanisms. While site-directed mutagenesis studies of G-protein coupled receptors have provided a host of inform ation regarding the domains essential for various functions, many mechanistic and structural questions remain to be resolved. We carried therefore carried out PCR-mediated random mutagenesis over a large part of the cAR1 sequence (from TMIII through the proximal part of the cytoplasmic tail). We devised a rapid screen for loss-of-function mutations based on the essential role of cAR1 of Dictyostelium in the developmental program. Although there were an average of two amino acid substitutions per receptor, ~90% of the mutants were able to substitute for wild-type cAR1 when expressed in receptor null cells. About 2% were loss-of-function mutants that expressed wild-type levels of receptor protein. We used biochemical screens to select about 100 of the se mutants and chose eight representative mutants for extensive characterization. These fell into distinct classes. One class had a conditional defect in cAMP binding that was reversed by high salt. Another large class had decreased affinity under all conditions. Curiously, the decreases were clustered into three discreet intervals. One of the most interesting class of mutants lost all capacity for signal transduction but were phosphorylated in response to agonist binding. This latter finding sugges ts that there are at least two activated states of cAR1 that can be recognized by different downstream effectors. --------------------------------------------------------------------- Subunit change in cytochrome c oxidase: identification of the oxygen switch in Dictyostelium Roberto Bisson, Silvia Vettore, Elisabetta Aratri and Dorianna Sandon Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via Trieste 75, 35121 Padova, Italy (email: bisson@civ.bio.unipd.it) EMBO J., in press Summary Cytochrome c oxidase (COX) has a complex modular structure in eukaryotes. Depending on growth conditions, interchangeable isoforms of selected subunits are synthesized and combined to the evolutionary-conserved catalytic core of the enzyme. In Dictyostelium this structural make-up is regulated by oxygen and involves two forms of the smallest subunit, termed VIIe and VIIs. Here we show that, in spite of a considerable sequence divergency, they are encoded by adjacent genes, linked "tail to head" by only 800 bp. Deletion analyses reveal the presence of a short intergenic segment acting as an oxygen transcriptional switch. This structural organization and the different stability of the two subunit isoforms offer a molecular explanation for the extraordinary sensitivity to oxygen of the switching mechanism. ---------------------------------------------------------------------- [End CSM-News, volume 7, number 11]