microscope. For the electron microscopy analysis, animals were fixed with glutaraldehyde and osmium tetraoxide (34). Five or six L4 or young adult animals were aligned within a small agar block, embedded, and sectioned together. Sections were poststained with uranyl acetate and lead citrate. 4. R. Durbin, thesis. University of Cambridge, Cambridge, England (1987). 5. C. Garriga, C. Desai, H. R. Horvitz. Development 117, 1071 (1993. 6. S. Kim and W. G. Wadsworth, data not shown. 7. The nid-7(RNAi) animals were generated as described previously [A. Fire et al., Nature 391. 806 (1998)l by using a 1-kb sequence from exon 8, which was cloned into pBluescript (Stratagene) as template for RNA synthesis. RNA was produced by both T3 and T7 RNA polymerase, and the reactions were pooled before being injected into the intestines of edls2O(unc-779::CFP) transgenic animals. Phenotypes of the nervous system were observed under epifluorescence microscopy. 8. Seven PCR fragments, including the whole coding sequence and intron region, were amplified from the genomic DNA of nid-l(ur47) animals. PCR fragments were cloned into a pBluescript vector and subsequently sequenced by automatic sequencer. The mutation was confirmed by sequencing two independent PCR fragments. . 16. lmmunostaining was performed by using freeze-fracture and methanol-acetone fixation as previously described (35). Polyclonal antibodies raised against mouse nidogen and a mouse monoclonal antibody against myosin heavy chain B (UNC-54) were used. For costaining, anti-rabbit fluorescein-conjugated and anti-mouse rhodamine-conjugated secondary antibodies were used. . 24. Transgenic strains were generated by standard methods (36). plM#194, an expression construct for nid-7, was constructed by cloning the 2.5-kb 5' flanking region of nid-7 into pPD 95.77 vector (from A. Fire). This CFP construct was coinjected at 10 pg/ml with pRF4 at 100 pg/ml. To establish a stable line, lM329 urls757 [plM#194, pRF4], transgenes were integrated by y-ray irradiation. For the ectopic expression construct of nid-7, constructs plM#195, plM#196, and plM#197, were made by using the 7-kb genomic nid-7 coding region, which was amplified by high-fidelity PCR, ligated to Nhe I-Bgl Il-digested vectors, pPD96.41, pPD49.83, and pPD96.52 (from A. Fire). These vectors contained 5' flanking regulatory sequences of mec-7, hsp76-47, and myo-3, respectively (36). The unc-779 regulatory sequence was amplified by using plM175 as template (23), and cloned into the pPD49.26 vector (from A. Fire) to construct plM#198. These constructs were injected at 10 pg/ml, with pRF4 into nid-l(ur47); kyls723 (zc27::CFP) animals. The resulting strains are IM330 urEx752 [plM#195]; nid-7 (ur47); kys123(zcZI::GFP); IM331 urEx753 [plM#196]; nid-l(ur47); kyls723 (zc27::tFP). lM332 urEx754 [plM#197]; nid-l(ur47); kyls723(zc27::CFP), 11' 1333 urEx755 [plM#198]; nidl(ur47); kyIs723(zc27::CFP). Ectopic expression of nid-7 was checked by in situ hybridization. IM331 emblyos collected 1 to 6 hours after being laid were heat-shock...

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo-and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.

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The lipocalin superfamily constitutes a phylogenetically conserved group of more than 40 proteins that function in the binding and transport of a variety of physiologically important ligands. Members of this family subserve diverse functions as carriers of retinoids (retinol binding protein), odorants (odorant binding proteins), chromophores (insecticyanin, INS), pheromones (aphrodisin) and sterols (apolipoprotein D, apoD). Despite the pivotal importance of the ligand binding function of these proteins, a suitable approach for characterizing the molecular determinants of such binding has not been available. In studies using three homogeneously purified lipocalins INS, beta-lactoglobulin (BLG) and human apoD, we find that the fluorescence reporter BIS (1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid) is an ideal candidate for use in rapid kinetic experiments and in fluorescence resonance energy transfer (FRET). These methods require only small amounts of reagents and yield molecular coordinates of the ligand binding cavity of lipocalins in solution that are in remarkably close agreement to those obtained from crystallographic work with solids. Extremely fast ligand binding dynamics is indicated.

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