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The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, also known as ErbB or HER, plays crucial roles in the development of multicellular organisms. Mutations and over-expression of the ErbB receptors have been implicated in a variety of human cancers. It is widely thought that the ErbB receptors are located in the plasma membrane, and that ligand binding to the monomeric form of the receptors induces its dimeric form for activation. However, it still remains controversial whether prior to ligand binding the receptors exist as monomers or dimers on the cell surface. Using bimolecular fluorescence complementation (BiFC) assays in the present study, we demonstrate that in the absence of bound ligand, all the ErbB family members have preformed, yet inactive, homo- and heterodimers on the cell surface, except for ErbB3 homodimers and heterodimers with cleavable ErbB4, which exist primarily in the nucleus. BiFC assays of the dimerization have also suggested that the ligand-independent dimerization of the ErbB receptors occurs in the endoplasmic reticulum (ER) before newly synthesized receptor molecules reach the cell surface. Based on BiFC and mammalian two-hybrid assays, it is apparent that the intracellular domains of the receptors are responsible for the spontaneous dimer formation. These provide new insights into an understanding of transmembrane signal transduction mediated by the ErbB family members, and are relevant to the development of anti-cancer drugs.

Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode Caenorhabditis elegans. Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner. These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system.In Caenorhabditis elegans, unc-13 mutants have uncoordinated movement and slow irregular pharyngeal pumping. There is evidence that the gene affects the nervous system rather than the musculature, which shows no visible defects. Abnormal connections between major interneurons through gap junctions have been found by reconstruction of part of the ventral nerve cord by serial section electron microscopy (I.N.M., unpublished results). It has been found by staining the nervous system with specific antibodies that motor and sensory neurons are misplaced in the unc-13 mutants (S. Siddiqui, personal communication). The mutants are also resistant to acetylcholinesterase inhibitors, such as aldicarb, but the enzyme is not altered (1). They also accumulate abnormally high levels of acetylcholine without alterations in the levels of choline or choline acetyltransferase activity (2).In this report, we describe the isolation and molecular characterization of the unc-13 gene* and show that it encodes a protein with a phorbol ester/diacylglycerol binding activity and a domain homologous to the regulatory region of protein kinase C (PKC), a multifunctional kinase that plays a central regulatory role in one of the major signal transduction pathways (3). MATERIALS AND METHODSNematode. The following C. elegans strains were used in this study. N2 is a standard wild-type; strain eSJ/e2274 is a partial revertant of the eSI mutant, e2153 and e2312 are weaker mutants spontaneously isolated from the background of a mutator strain, TR679 (4), and eJO91 is an amber mutant isolated from N2 after ethyl methanesulfonate mutagenesis. The conditions for growth, maintenance, and genetic manipulation of the nematode have been described (5, 6).DNA Procedures. Restriction enzymes, T4 DNA ligase, and Escherichia coli DNA polymerase Klenow fragment were used under the standard conditions (7). DNA sequencing was carried out by the chain-termination method (8) after cloning relevant fragments into M13 vectors (9). Oligonucleotides were synthesized by a DNA synthesizer (model 380B, Applied Biosystems).A genomic A library was made with A2001 (10) from N2 genomic DNA by partial digestion with Sau3A1 restriction enzyme, followed by purification of 15-to 20-kilobase (kb) fragments in an ag...

Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS), introduced to study biomolecular interactions, has recently been reported to monitor enzyme activity by using a newly developed fluorescent protein variant together with cyan fluorescent protein. Here, for the first time to our knowledge, SW-FCCS is applied to detect interactions between membrane receptors in vivo by using the widely used enhanced green fluorescent protein and monomeric red fluorescent protein. The biological system studied here is the epidermal growth factor/ErbB receptor family, which plays pivotal roles in the development of organisms ranging from worms to humans. It is widely thought that a ligand binds to the monomeric form of the receptor and induces its dimeric form for activation. By using SW-FCCS and Förster resonance energy transfer, we show that the epidermal growth factor receptor and ErbB2 have preformed homo- and heterodimeric structures on the cell surface and quantitation of dimer fractions is performed by SW-FCCS. These receptors are major targets of anti-cancer drug development, and the receptors' homo- and heterodimeric structures are relevant for such developments.