Synthesis of determinant oligosaccharides of the ABH (type 1) blood group antigen and Leb tetrasaccharide from a common precursor

Synthesis of blood group ABH (type 1) determinant oligosaccharides and Leb tetrasaccharide has been performed using the same trisaccharide precursor-benzyl 2-acetamido-4,6-O-benzylidene-[4,6-O-benzylidene-2-O-[2-O-benzyl-3,4-di- O- (4-nitrobenzoyl)-alpha-L-fucopyranosyl]-beta-D-galactopyranosyl]-2-deoxy - alpha-D-glucopyranoside. A- and B-determinants were prepared by alpha-galactosaminylation and alpha-galactosylation of the title trisaccharide, respectively. Leb-determinant was synthesized by a series of simple blocking and deblocking steps followed by alpha-fucosylation.     

Synthesis of oligosaccharides with group specificity for blood types A, B and H (Type 3)

Chemical synthesis of A, B, and H (type 3) human blood group determinant oligosaccharides (as R-glycosides, R = OCH2CH2CH2NHCOCF3) and their polymeric derivatives are reported. 4,6; 4',6'-Di-O-benzylidene derivative of Gal beta 1----3GalNAc alpha 1----R was chloroacetylated selectively at 3'-OH, the chloroacetate was alpha-fucosylated and dechloroacetylated to give protected H (type 3) trisaccharide bearing free 3'-OH. alpha-Glycosylation of the trisaccharide with 2-azido-3,4,6-tri-O-benzyl-beta-D-galactopyranosyl chloride and 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl bromide gave rise to protected A and B tetrasaccharides, respectively. Deprotected R-glycosides were converted to OCH2CH2CH2NH2 derivatives. Their reaction with poly(4-nitrophenylacrylate) affords polyacrylamide-coupled conjugates with A, B, and H (type 3) specificity.     

Ligand-binding properties of annexin from Caenorhabditis elegans (annexin XVI, Nex-1)

Annexins are structurally related proteins that bind phospholipids in a calcium-dependent manner. Recently, we showed that annexins IV, V, and VI also bind glycosaminoglycans in a calcium-dependent manner. Annexins are widely distributed from lower to higher eukaryotes, and the nematode Caenorhabditis elegans has been found to contain Nex-1, an annexin homologue. Here, we characterize the ligand-binding properties of Nex-1 using recombinant Nex-1. Nex-1 binds to liposomes containing phosphatidylserine. The apparent K(d) was calculated by Biacore to be 4.4 nM. Compared to mammalian annexins, the Nex-1 phospholipid-binding specificities were similar whereas the K(d) values were one order of magnitude larger. The Nex-1 glycosaminoglycan-binding specificities were investigated by affinity chromatography and solid-phase assays. Nex-1 binds to heparin, heparan sulfate, and chondroitin sulfate but not to chondroitin and chemically N- or O-desulfated heparin. Besides phospholipids, heparan sulfate and/or chondroitin (sulfate), probably on perlecan, could be endogenous ligands of Nex-1.     

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background

Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages.

Results

We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers.

Conclusions

Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.     

Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.     

Sugar binding properties of the two lectin domains of the tandem repeat-type galectin LEC-1 (N32) of Caenorhabditis elegans. Detailed analysis by an improved frontal affinity chromatography method

The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing N-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Galbeta1-3GlcNAc to Galbeta1-4GlcNAc. Because the Fucalpha1-2Galbeta1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the K(d) value of pentasaccharide Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 x 10(-5), 1.3 x 10(-4), and 6.5 x 10(-5) m, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA, which contains the blood group A antigen; the K(d) value for immobilized Nh was 4.8 x 10(-5) m, and that for Ch was 8.1 x 10(-4) m. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.     

Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.     

Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3

Galectins are a growing family of animal lectins with common consensus sequences that bind beta-Gal and LacNAc residues. There are at present 14 members of the galectin family; however, certain galectins possess different structures as well as biological properties. Galectin-1 is a dimer of two homologous carbohydrate recognition domains (CRDs) and possesses apoptotic and proinvasive activities. Galectin-3 consists of a C-terminal CRD and an N-terminal nonlectin domain implicated in the oligomerization of the protein and is often associated with antiapoptotic activity. Because many cellular oligosaccharide receptors are multivalent, it is important to characterize the interactions of multivalent carbohydrates with galectins-1 and -3. In the present study, binding of bovine heart galectin-1 and recombinant murine galectin-3 to a series of synthetic analogs containing two LacNAc residues separated by a varying number of methylene groups, as well as biantennary analogs possessing two LacNAc residues, were examined using isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements. The thermodynamics of binding of the multivalent carbohydrates to the C-terminal CRD domain of galectin-3 was also investigated. ITC results showed that each bivalent analog bound by both LacNAc residues to the two galectins. However, galectin-1 shows a lack of enhanced affinity for the bivalent straight chain and branched chain analogs, whereas galectin-3 shows enhanced affinity for only lacto-N-hexaose, a naturally occurring branched chain carbohydrate. The CRD domain of galectin-3 was shown to possess similar thermodynamic binding properties as the intact molecule. The results of this study have important implications for the design of carbohydrate inhibitors of the two galectins.     

Monoclonal antibodies defining blood group A variants with difucosyl type 1 chain (ALeb) and difucosyl type 2 chain (ALey)

Three hybridomas secreting monoclonal antibodies, HH1, HH2, and HH3, defining different difucosyl A structures (ALeb or ALey), have been established. Antibody HH1 (IgG2a) reacts specifically with the difucosyl A structure irrespective of a type 1 or type 2 chain, while antibody HH2 (IgG3) reacts exclusively with the difucosyl type 2 chain A (ALey) and does not react with the difucosyl type 1 chain or monofucosyl type 2 chain. Antibody HH3 (IgG2a) reacts exclusively with the difucosyl type 1 chain A (ALeb) and does not react with the monofucosyl type 1 chain A or mono- and difucosyl type 2 chain A. These hybridoma antibodies were obtained by immunization of mice with purified glycolipid antigens and were selected by their reactivity with the specific glycolipid structures. These antibodies, together with previously established monoclonal antibody AH-21, specific for monofucosyl type 1 chain A, and monoclonal antibody TH-1, specific for type 3 chain A, are extremely useful to define blood group A variants present in cells and tissues.     

Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span

We have cloned and functionally characterized two Na(+)-coupled dicarboxylate transporters, namely ceNaDC1 and ceNaDC2, from Caenorhabditis elegans. These two transporters show significant sequence homology with the product of the Indy gene identified in Drosophila melanogaster and with the Na(+)-coupled dicarboxylate transporters NaDC1 and NaDC3 identified in mammals. In a mammalian cell heterologous expression system, the cloned ceNaDC1 and ceNaDC2 mediate Na(+)-coupled transport of various dicarboxylates. With succinate as the substrate, ceNaDC1 exhibits much lower affinity compared with ceNaDC2. Thus, ceNaDC1 and ceNaDC2 correspond at the functional level to the mammalian NaDC1 and NaDC3, respectively. The nadc1 and nadc2 genes are not expressed at the embryonic stage, but the expression is detectable all through the early larva stage to the adult stage. Tissue-specific expression pattern studies using a reporter gene fusion approach in transgenic C. elegans show that both genes are coexpressed in the intestinal tract, an organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Independent knockdown of the function of these two transporters in C. elegans using the strategy of RNA interference suggests that NaDC1 is not associated with the regulation of average life span in this organism, whereas the knockdown of NaDC2 function leads to a significant increase in the average life span. Disruption of the function of the high affinity Na(+)-coupled dicarboxylate transporter NaDC2 in C. elegans may lead to decreased availability of dicarboxylates for cellular production of metabolic energy, thus creating a biological state similar to that of caloric restriction, and consequently leading to life span extension.     

The serotonin receptor SER-1 (5HT2ce) contributes to the regulation of locomotion in Caenorhabditis elegans

Serotonin (5-hydroxytryptamine: 5HT) is an important neuroactive substance in the model roundworm, Caenorhabditis elegans. Aside from having effects in feeding and egg-laying, 5HT inhibits motility and also modulates several locomotory behaviors, notably food-induced slowing and foraging. Recent evidence showed that a serotonergic 5HT2-like receptor named SER-1 (also known as 5HT2ce) was responsible for the effect of 5HT on egg-laying. Here we confirm this observation and show that SER-1 also plays an important role in locomotion. A mutant lacking SER-1 was found to be highly resistant to exogenous 5HT in the absence of food and this resistant phenotype was rescued by reintroducing the SER-1 gene in a mutant background. Pharmacological studies showed that the same antagonists that blocked the activity of recombinant SER-1 in vitro also inhibited the effect of 5HT on motility, suggesting the same receptor was responsible for both effects. When tested for locomotory behaviors, the SER-1 mutant was found to be moderately defective in food-induced slowing. In addition, the mutant changed direction more frequently than the wildtype when searching for food, suggesting that SER-1 may play a role in navigational control during foraging. Both these effects required the presence of MOD-1, a 5HT gated chloride channel, and the results indicate that SER-1 and MOD-1 modulate these behaviors through a common pathway. On the basis of expression analysis of a ser-1::GFP translational fusion, SER-1 is prominently located in central, integrating neurons of the head ganglia (RIA and RIC) but not the body wall musculature. The evidence suggests that SER-1 controls locomotion through indirect modulation of neuromuscular circuits and has effects both on speed and direction of movement.     

Characterization of blood group ABO(H)-active gangliosides in type AB erythrocytes and structural analysis of type A-active ganglioside variants in type A human erythrocytes

Several monosialogangliosides containing the type A-active epitope have been detected in type A erythrocytes on immunological analysis with a monoclonal antibody, and three of them were purified by repeated silica bead column chromatography and by scraping from the TLC plate. Two of these A-active gangliosides were characterized by methylation analysis by GC/MS, negative SIMS, MALDI-TOF/MS, proton nuclear magnetic resonance spectroscopy, and immunological assays, and their structures were concluded to be as follows. A-active ganglioside I:A-active ganglioside II:The reactivity of the purified gangliosides to the anti-A monoclonal antibodies (mAbs) exhibited enhancement after removal of the sialic acid. Therefore, the sialic residue has been shown to inhibit the binding to the terminal A-active epitope through the formation of an immune complex. To confirm the presence of A- (including S-A-I, -II and -III) and B-active gangliosides, the reactivity of anti-A and -B mAbs were investigated using total gangliosides from type A, -B and -AB erythrocytes on TLC plate. The results were that the gangliosides from types A and AB showed positive reaction to anti-A mAbs, whereas in the anti-B mAbs binding the gangliosides from types B and AB were positive. Thus, it revealed that A-active gangliosides were present in type A and -AB, and B-active gangliosides in types B and AB. As there was no difference in respective gangliosides on type AB erythrocytes of 22 individuals, both A- and B-active gangliosides are equally present in type AB erythrocytes. The biological significance of these A- and B-active ganglioside variants remains vague at present. As these molecules exhibit different reactivities to the anti-A mAbs, it is very likely that they can regulate the antigenicity of the A-epitope on the cell surface.     

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.     

Cloning within the unc-43 to unc-31 interval (linkage group IV) of the Caenorhabditis elegans genome using Tc1 linkage selection

The region around the twitcher gene, unc-22, flanked by unc-43 on the left and by unc-31 on the right, has been intensively studied in our laboratory over the period of the last 8 years. In this paper we describe the identification and isolation of probes specific for several restriction fragment length differences (RFLDs) which lie within this region. Many RFLDs in Caenorhabditis elegans are caused by the insertion of a transposable element, Tc1. The method we used involved the isolation of Tc1-containing genomic fragments. These were recovered from a lambda gt 10 library of DNA from a specially constructed genetic strain containing the unc-43 to unc-31 interval from the BO strain and the rest of the genome from N2. Because the BO strain is rich in Tc1 insertion sites and the N2 strain has few, the majority of Tc1-bearing genomic fragments in the constructed strain were derived from the unc-22 region. Of nine such Tc1-bearing genomic fragments isolated, six were found which mapped within the region of interest. The 350 kilobases of genomic sequences isolated as a result of these studies are being used to study the molecular organization of this region. The method described here for Tc1 linkage selection is one that is rapid, general, and may be targeted to any genetically characterized region of the C. elegans genome.     

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.     

Novel monoclonal antibodies with specificity for chelated uranium(VI): isolation and binding properties

A derivative of 1,10-phenanthroline that binds to UO(2)(2+) with nanomolar affinity was found to be a very effective immunogen for the generation of antibodies directed toward chelated complexes of hexavalent uranium. This study describes the synthesis of 5-isothiocyanato-1,10-phenanthroline-2,9-dicarboxylic acid and its use in the generation and functional characterization of a group of monoclonal antibodies that recognize the most soluble and toxic form of uranium, the hexavalent uranyl ion (UO(2)(2+)). Three different monoclonal antibodies (8A11, 10A3, and 12F6) that recognize the 1:1 complex between UO(2)(2+) and 2,9-dicarboxy-1,10-phenanthroline (DCP) were produced by the injection of BALB/c mice with DCP-UO(2)(2+) covalently coupled to a carrier protein. Equilibrium dissociation constants for the binding of DCP-UO(2)(2+) to antibodies 8A11, 10A3, and 12F6 were 5.5, 2.4, and 0.9 nM, respectively. All three antibodies bound the metal-free DCP with roughly 1000-fold lower affinity. The second-order rate constants for the bimolecular association of each antibody with soluble DCP-UO(2)(2+) were in the range of 1 to 2 x 10(7) M(-1) s(-1). Binding studies conducted with structurally related chelators and 21 metal ions demonstrated that each of these three antibodies was highly specific for the soluble DCP-UO(2)(2+) complex. Detailed equilibrium binding studies conducted with three other derivatives of DCP, either complexed with UO(2)(2+) or metal-free, suggested that the antigen binding sites on the three antibodies have significant functional and structural similarities. Biomolecules that bind specifically to uranium will be at the heart of any new biotechnology developed to monitor and control uranium contamination. The three antibodies described herein possess sufficient affinity and specificity to support the development of immunoassays for hexavalent uranium in environmental and clinical samples.     

SER-1, a Caenorhabditis elegans 5-HT2-like receptor, and a multi-PDZ domain containing protein (MPZ-1) interact in vulval muscle to facilitate serotonin-stimulated egg-laying

Serotonin (5-HT) stimulation of egg-laying in Caenorhabditis elegans is abolished in ser-1 (ok345) animals and is rescued by ser-1 expression in vulval muscle. A PDZ binding motif (ETFL) at the SER-1 C-terminus is not essential for rescue, but facilitates SER-1 signaling. SER-1 binds specifically to PDZ domain 10 of the multi-PDZ domain protein, MPZ-1, based on GST pulldown and co-immunoprecipitation. mpz-1 is expressed in about 60 neurons and body wall and vulval muscles. In neurons, GFP-tagged MPZ-1 is punctate and colocalizes with the synaptic marker, synaptobrevin. The expression patterns of ser-1 and mpz-1 overlap in 3 pairs of neurons and vulval muscle. In addition, MPZ-1 also interacts with other GPCRs with acidic amino acids in the -3 position of their PDZ binding motifs. mpz-1 RNAi reduces 5-HT stimulated egg-laying in wild type animals and in ser-1 mutants rescued by muscle expression of SER-1. In contrast, mpz-1 RNAi has no effect on 5-HT stimulated egg-laying in ser-1 mutants rescued by expression of a truncated SER-1 that lacks the C-terminal PDZ binding motif. The overexpression of MPZ-1 PDZ domain 10 also inhibits 5-HT stimulated egg-laying. These studies suggest that the SER-1/MPZ-1 interaction facilitates SER-1 mediated signaling.