[
European Worm Meeting,
2000]
Guanine nucleotide exchange factors (GEFs) are proteins that activate members of the Ras-family of small GTPases following stimulation of cell surface receptors. Recently, various novel, Rap1-specific GEFs have been cloned. They all have a highly homologous catalytic domain as well as various other domains, which may regulate catalytic activity and/or determine their subcellular location. For example, PDZ-GEF1(also named RA-GEF or nRap GEP) is characterized by the presence of a PDZ-domain, a Ras-binding domain, and a domain that resembles a cyclic nucleotide-binding domain, yet cannot bind cAMP nor cGMP. We have cloned the C. eleganshomolog of PDZ-GEF1 (T14G10.2) and show here that like its mammalian counterpart is also a Rap1-specific exchange factor. Using a GFP fusion construct we find that it becomes expressed first during embryogenesis in certain neuronal cells, and slightly later in hypodermal cells. At later stages expression is found in the neurons in the head and tail, in the pharynx, in the sheath cells of the uterus and in the distal tip cell. We identified worms carrying a mutation in cePDZ-GEF1, that deletes part of the PDZ-domain and the complete catalytic domain. Therefore, it is likely that this allele represents a null allele. Worms homozygous for this mutation hatch normally but remain smaller and develop slower as compared to wild type animals. They move in an uncoordinated fashion and have severe problems in moulting. Large vesicles are frequently seen in various parts of the body and the gut seems enlarged. Most animals die before adulthood, whereas those that reach this stage are largely sterile. Mutant animals can be rescued by injection with a cosmid containing the cePDZ-GEF1 gene. Together, these data show that cePDZ-GEF1 is an essential gene, expressed in a tissue-specific fashion.
[
European Worm Meeting,
2000]
In vertebrate cells activation of the small Ras-like GTPase Rap1 by the second messenger cAMP is mediated by the guanine nucleotide exchange factors EPAC (Exchange Protein Activated by cAMP) and EPAC2. The genome of C. eleganscontains a single homologue of EPAC that contains two cAMP-binding motifs, which may regulate the activity of the catalytic domain at the C-terminus. In addition, it contains a DEP domain, most likely involved in membrane targeting and a REM domain, which is found in many nucleotide exchange factors, but whose function has not been defined. Biochemical characterization of the protein encoded by this gene shows that it also is a Rap-specific exchange factor. Using a GFP-reporter construct we show that EPAC is expressed in various neurons in the head as well as in neurons in the ventral nerve cord. Also expression in the pharynx is seen. In order to learn more about the function of EPAC we attempted to identify animals carrying mutated versions of this gene. A single mutation was found in which most of the first and all of the second cAMP-binding motif as well as the REM and DEP domains are deleted. It may be expected that the protein produced from this allele is no longer responsive to cAMP and is largely cytoplasmic because of the absence of the DEP domain. At least a fraction of animals homozygous for this allele is viable and fertile. We are presently in the process of analysing the mutant phenotype.