Scope and Method of Study. The Dictyostelium G protein subunit, Gα5, 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 Gα5 subunit was created with alterations in the D-motif (Gα5^d-) and compared to the wild-type subunit (Gα5). Because the Dictyostelium genome encodes two MAPKs, Erk1 and Erk2, interactions of these proteins with Gα5 and Gα5^d- were assayed to understand roles of the D-motif.

Findings and Conclusions. Over-expression of Gα5 was lethal to vegetative cells, but cells over-expressing Gα5 ^d- maintained normal growth suggesting the Gα5-associated lethality is regulated by the D-motif. Unlike Gα5 over-expression, Gα5 ^d- expressed in gα5- cells was unable to reduce the large aggregate size associated with gα5- cell development suggesting the D-motif regulates aggregate size. Over-expression of Gα5^d- did not provide accelerated prestalk gene expression or tip formation as observed for Gα5 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 Gα5 mutants in a yeast two-hybrid analysis indicated interaction of the MAPK with Gα proteins possessing and lacking the D-motif. Western blotting indicated phosphorylation of the MAPKs was similar in gα5- cells expressing either Gα5 or Gα5^d- 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 Gα subunit with (Gα5*) or without (Gα5^d-, *) the D-motif suggested Gα5* dissociates from Erk2 after cells are stimulated with folic acid. Additional Western blotting indicated that another Dictyostelium Gα subunit, Gα4, might be required for activation of both MAPKs.

Additional aspects of G protein-mediated signaling were also explored. Analysis of GrlH (GABA_B 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.