Although many positive components of the LET-23 mediated signaling pathway have been identified and characterized, negative regulation of this pathway is less understood. Several negative regulators have been identified including
sli-1,
sli-2,
rok-1,
gap-1 and
unc-101. A mutation in any one of these negative regulators results in a wild-type vulva. However, certain double mutant combinations of these negative regulators results in a Multivulva phenotype. We are currently characterizing the negative regulator
sli-2.
sli-2 was originally isolated as a semi-dominant suppressor of the reduction of function receptor allele
let-23(
sy1).
sli-2 appears to act upstream of
let-60 RAS and downstream or in parallel with
let-23.
sli-2 maps to chromosome V between
unc-46 and
dpy-11. We are currently attempting to clone
sli-2. Another negative regulator,
unc-101, encodes the medium chain subunit of the trans-Golgi clathrin adaptor complex. We are interested in how a gene presumably affecting protein sorting interacts with the LET-23 mediated signaling pathway. It appears that
unc-101 is not acting by simply increasing receptor in the vulva precursor cells based on the following evidence: Overexpression of LET-23 receptor does not phenocopy the
unc-101 vulval defect. There appears to be no detectable difference in LET-23::GFP localization in
unc-101 mutants. We have detected no interaction of LET-23 receptor with components of the trans-Golgi clathrin adaptor complex using the yeast two-hybrid assay. We are performing a screen for suppressors of
unc-101 to help determine the mechanism by which
unc-101 acts. * Supported by the U.S. Army Breast Cancer Research Program.