| Scope and Method of Study. The Dictyostelium G protein subunit, Galpha5, has been shown to possess a putative D-motif or mitogen-activated protein kinase (MAPK) docking site. To analyze the function of this D-motif, a mutant Galpha5 subunit was created with alterations in the D-motif (Galpha5d-) and compared to the wild-type subunit (Galpha5). Because the Dictyostelium genome encodes two MAPKs, Erk1 and Erk2, interactions of these proteins with Galpha5 and Galpha5d- were assayed to understand roles of the D-motif.;Findings and Conclusions. Over-expression of Galpha5 was lethal to vegetative cells, but cells over-expressing Galpha5 d- maintained normal growth suggesting the Galpha5-associated lethality is regulated by the D-motif. Unlike Galpha5 over-expression, Galpha5 d- expressed in galpha5- cells was unable to reduce the large aggregate size associated with galpha5- cell development suggesting the D-motif regulates aggregate size. Over-expression of Galpha5d- did not provide accelerated prestalk gene expression or tip formation as observed for Galpha5 suggesting the D-motif is required for these developmental processes. Co-expression of a truncated Erk2 (Erk2CT) containing the CD-motif of the MAPK and Galpha5 mutants in a yeast two-hybrid analysis indicated interaction of the MAPK with Galpha proteins possessing and lacking the D-motif. Western blotting indicated phosphorylation of the MAPKs was similar in galpha5- cells expressing either Galpha5 or Galpha5d- subunits and stimulated with folic acid. Western blotting following cobalt resin pull-down assays using lysates from folate-stimulated cells expressing His6-Erk2 and Myc-tagged forms of a constitutively active Galpha subunit with (Galpha5*) or without (Galpha5d-, *) the D-motif suggested Galpha5* dissociates from Erk2 after cells are stimulated with folic acid. Additional Western blotting indicated that another Dictyostelium Galpha subunit, Galpha4, might be required for activation of both MAPKs.;Additional aspects of G protein-mediated signaling were also explored. Analysis of GrlH (GABAB receptor- like H) provided no vegetative or developmental phenotypes, but additional work could potentially elucidate a function for GrlH. Development of a biological sensor utilizing the natural ability of Dictyostelium to sense folic acid was also attempted. While this project was not completed, several DNA constructs were created that could be of use to the Dictyostelium community. |
