A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.

Positive and negative tissue-specific signaling by a nematode epidermal growth factor receptor.

The major determinants of receptor tissue tyrosine kinase (RTK) signaling specificity have been proposed to be Src homology 2 (SH2) binding sites, phosphotyrosine-containing oligopeptides in the cytoplasmic domain of the receptor. The Caenorhabditis elegans epidermal growth factor receptor homologue LET-23 has multiple functions during development and has eight potential SH2-binding sites in a region carboxyl terminal to its kinase domain. By analyzing transgenic nematodes for three distinct LET-23 functions, we show that six of eight potential sites function in vivo and that they are required for most, but not all, of LET-23 activity. A single site is necessary and sufficient to promote wild-type fertility. Three other sites activate the RAS pathway and are involved only in viability and vulval differentiation. A fifth site is promiscuous and can mediate all three LET-23 functions. An additional site mediates tissue-specific negative regulation. Putative SH2 binding sites are thus key effectors of both cell-specific and negative regulation in an intact organism. We suggest two distinct mechanisms for tissue-specific RTK-mediated signaling. A positive mechanism would promote RTK function through effectors present only in certain cell types. A negative mechanism would inhibit RTK function through tissue-specific negative regulators.

Requirements of multiple domains of SLI-1, a Caenorhabditis elegans homologue of c-Cbl, and an inhibitory tyrosine in LET-23 in regulating vulval differentiation.

SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin-protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss of sli-1 but not of another negative regulator, ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin-ligase domain.

Polyunsaturated fatty acids and neurotransmission in Caenorhabditis elegans.

Changes in PUFA (polyunsaturated fatty acid) metabolism can cause mental retardation and cognitive impairment. However, it is still unclear why altered levels of PUFAs result in neuronal dysfunction. Recent studies on the nematode Caenorhabditis elegans suggest that PUFA depletion may cause cognitive impairment by compromising communication among neurons. Pharmacological and electrophysiological experiments showed that animals devoid of most PUFAs release abnormally low levels of neurotransmitters. In addition, ultrastructural analysis revealed that synapses in these mutants are severely depleted of synaptic vesicles. The conclusion of these studies is that PUFAs are required to maintain a normal pool of synaptic vesicles at pre-synaptic sites, thus ensuring efficient neurotransmission.

Mutations in the Caenorhabditis elegans let-23 EGFR-like gene define elements important for cell-type specificity and function.

The Caenorhabditis elegans let-23 gene is a genetically characterized member of the epidermal growth factor receptor (EGFR) tyrosine kinase family. Mutations in let-23 can produce five phenotypes in the nematode. Alleles of let-23 include null alleles, reduction-of-function alleles and alleles that disrupt function in some cell types and not others. We have sequenced some of these mutations to identify sequences and regions important for overall let-23 function and for let-23 function in specific cell types. Our data indicate that in vivo, the receptor's C-terminus can be partitioned into at least three domains that each contribute to receptor function in different cell types. In particular, we find distinct domains that mediate hermaphrodite fertility and vulval induction. Our data also demonstrate for the first time that a single, conserved residue in the ligand binding domain is critical for function in vivo and that mutations in the extracellular cysteines characteristic of the EGFR family can lead to a partial or a complete reduction of receptor function.

The amino-terminal domain of the golgi protein giantin interacts directly with the vesicle-tethering protein p115.

Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat protomer I vesicles to Golgi membranes and the initial stacking of reforming cisternae. Here we show that the NH(2)-terminal 15% of Giantin suffices to bind p115 in vitro and in vivo and to block cell-free Golgi reassembly. Because Giantin is a long, rod-like protein anchored to the membrane by its extreme COOH terminus, these results support the idea of a long, flexible tether linking vesicles and cisternae.