ABSTRACT
Wnt glycoproteins have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize beta-catenin. Wnt signalling is antagonized by the extracellular Wnt inhibitor dickkopf1 (dkk1), which is a member of a multigene family. dkk1 was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks Wnt signalling is unknown. LDL-receptor-related protein 6 (LRP6) is required during Wnt/beta-catenin signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for Wnt. Here we show that LRP6 (ref. 7) is a specific, high-affinity receptor for Dkk1 and Dkk2. Dkk1 blocks LRP6-mediated Wnt/beta-catenin signalling by interacting with domains that are distinct from those required for Wnt/Frizzled interaction. dkk1 and LRP6 interact antagonistically during embryonic head induction in Xenopus where LRP6 promotes the posteriorizing role of Wnt/beta-catenin signalling. Thus, DKKs inhibit Wnt co-receptor function, exemplifying the modulation of LRP signalling by antagonists.
Subject(s)
Proteins/metabolism , Receptors, Immunologic/metabolism , Receptors, LDL/metabolism , Trans-Activators , Xenopus Proteins , Zebrafish Proteins , Adaptor Proteins, Signal Transducing , Animals , Binding Sites , Cell Line , Chemokines , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Embryonic Induction , Head/embryology , Humans , Intercellular Signaling Peptides and Proteins , Low Density Lipoprotein Receptor-Related Protein-1 , Low Density Lipoprotein Receptor-Related Protein-6 , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Proteins/antagonists & inhibitors , Proteins/chemistry , Proteins/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, LDL/antagonists & inhibitors , Receptors, LDL/chemistry , Receptors, LDL/genetics , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/metabolism , Sequence Deletion/genetics , Signal Transduction , Substrate Specificity , Wnt Proteins , Xenopus laevis/embryology , beta CateninABSTRACT
Formyl-methionine-containing peptides (e.g. fMet-Leu-Phe) stimulate a variety of neutrophil functions by interacting with specific cell surface receptors which are coupled via G-proteins to stimulation of phospholipase C. Two markedly distinct cDNAs coding for formyl peptide receptors have recently been isolated from a rabbit and a human cDNA library respectively. To examine the hitherto unknown signal transduction properties of the formyl peptide receptor encoded by the human cDNA, we have used the PCR to clone this cDNA from poly(A)+ RNA of myeloid differentiated human leukaemia (HL-60) cells, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes. Receptor activity was determined electrophysiologically by measuring the agonist-dependent opening of intracellular Ca2+ concentration ([Ca2+]i)-independent Cl- channels. Injection of pure formyl peptide receptor cRNA did not lead to peptide-dependent changes in membrane current. In contrast, marked alterations of membrane current were observed in response to formyl peptides when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells. Injection of the latter RNA did not lead to formyl-peptide-dependent alterations of membrane current. Binding studies using a radioiodinated formyl peptide revealed that injection of formyl peptide receptor cRNA alone led to expression of the formyl peptide receptor on the oocyte surface, and that co-injection of poly(A)+ RNA from undifferentiated HL-60 cells did not alter the level of receptor expression. Size fractionation of poly(A)+ RNA from undifferentiated HL-60 cells showed that the mRNA required to complement formyl-peptide-dependent signal transduction in oocytes had a size of approx. 3-3.5 kb. These results strongly suggest that the human formyl peptide receptor requires a specific cofactor(s), which is lacking in Xenopus oocytes but is present in undifferentiated HL-60 cells, to activate the second messenger pathway in oocytes. Identification of this factor will provide important information about the molecular mechanisms by which G-protein-coupled granulocyte-activating receptors stimulate phospholipase C.
Subject(s)
DNA/genetics , Oocytes/physiology , Receptors, Immunologic/genetics , Signal Transduction/genetics , Xenopus laevis/physiology , Amino Acid Sequence , Animals , Base Sequence , Female , Humans , Leukemia, Experimental/genetics , Leukemia, Experimental/pathology , Leukemia, Myeloid/genetics , Leukemia, Myeloid/pathology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Polymerase Chain Reaction , RNA, Messenger/genetics , Receptors, Formyl Peptide , Receptors, Immunologic/physiology , Signal Transduction/physiology , Tumor Cells, Cultured , Xenopus laevis/geneticsABSTRACT
We have used the polymerase chain reaction to isolate and clone the cDNA encoding the human C5a receptor, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes for functional characterization of the receptor protein. Receptor activity was determined either electrophysiologically by measuring the agonist-dependent opening of [Ca2+]i-dependent Cl- channels, or by analysing the agonist-dependent efflux of 45Ca2+ from the oocytes. Using both methodologies, injection of pure C5a receptor cRNA failed to confer C5a sensitivity on the oocytes. In contrast, marked responses to C5a were observed when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells, which is devoid of C5a receptor mRNA. Binding studies using radioiodinated C5a revealed that the C5a receptor polypeptide was in fact synthesized and targeted to the oocyte plasma membrane in oocytes injected with receptor cRNA alone, and that the level of receptor expression was not influenced by coinjection of poly(A)+ RNA from undifferentiated HL-60 cells. These results strongly suggest that the human C5a receptor requires a specific cofactor(s) lacking in Xenopus oocytes but present in undifferentiated HL-60 cells, to generate intracellular signals in oocytes. Identification and characterization of this factor will provide important information about the molecular mechanisms by which G-protein-coupled receptors activate phospholipase C.
