dictyNews Electronic Edition Volume 28, number 16 June 8, 2007 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Regulation of G-protein-coupled cAMP receptor activation by a hydrophobic residue in transmembrane helix 3 Minghang Zhang, Mousumi Goswami, Satoshi Sawai, Edward C. Cox, and Dale Hereld Molecular Microbiology, in press Summary: cAR1, a G-protein-coupled cAMP receptor, is essential for multicellular development of Dictyostelium. We previously identified a cAR1-Ile104 mutant that appeared to be constitutively activated based on its constitutive phosphorylation, elevated affinity for cAMP, and dominant-negative effects on development as well as specific cAR1 pathways that are subject to adaptation. To investigate how Ile104 might regulate cAR1 activation, we assessed the consequences of substituting it with all other amino acids. Constitutive phosphorylation of these Ile104 mutants varied broadly, suggesting that they are activated to varying extents, and was correlated with polarity of the substituting amino acid residue. Remarkably, all Ile104 substitutions, except for the most conservative, dramatically elevated the receptor's cAMP affinity. However, only a third of the mutants (those with the most polar substitutions) blocked development. These findings are consistent with a model in which polar Ile104 substitutions perturb the equilibrium between inactive and active cAR1 conformations in favor of the latter. Based on homology with rhodopsin, Ile104 is likely buried within inactive cAR1 and exposed to the cytoplasm upon activation. We propose that the hydrophobic effect normally promotes burial of Ile104 and hence cAR1 inactivation, while polar substitution of Ile104 mitigates this effect, resulting in activation. Submitted by: Dale Hereld [hereldd@niaid.nih.gov] -------------------------------------------------------------------------------- GxcDD, a putative RacGEF, is involved in Dictyostelium development Subhanjan Mondal, Dhamodharan Neelamegan, Francisco Rivero, Angelika. A. Noegel Institute for Biochemistry I, Medical Faculty, and Center for Molecular Medicine, University of Cologne, 50931 Cologne BMC Cell Biology Background Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor. Results We report on GxcDD (Guanine exchange factor for Rac GTPases), a Dictyostelium RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling. Conclusions The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in Dictyostelium, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further. Submitted by: Angelika. A. Noegel [noegel@uni-koeln.de] ============================================================== [End dictyNews, volume 28, number 16]