b-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous b-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N-and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant b-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/b-catenin signaling in dorsal neural tube development. While loss of b-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/b-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of b-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity.[Keywords: canonical Wnt signaling; signaling versus structural function of b-catenin; mouse strains expressing transcriptionally inactive b-catenin; cell fate determination in the dorsal neural tube]

Three receptor-activity-modifying proteins (RAMP) define specific interactions between calcitonin (CT) gene-related peptide (CGRP), adrenomedullin (AM) and amylin, and a CT receptor or a CT receptor-like receptor (CRLR). Both form heterodimeric RAMP/receptor complexes at the cell surface. This association represents a novel principle of G protein-coupled receptor function. RAMP1 is transported to the cell surface together with the CRLR or the CT receptor. Here, we have investigated the functional relevance of the short C-terminal intracellular tail QSKRTEGIV and of the single transmembrane domain of human (h) RAMP1 for their interactions with the hCRLR to constitute a CGRP receptor. To this end, hRAMP1 has been sequentially truncated from the C-terminus, and [125I]hαCGRP/hRAMP1/hCRLR association at the cell surface and cAMP accumulation in response to hαCGRP have been examined. With the C-terminal truncation of hRAMP1 by four amino acids wild-type hRAMP1 function was maintained, and the hCRLR was required for the transport of hRAMP1 to the cell surface. Further truncation of hRAMP1 through removal of the remaining five intracellular amino acids revealed CRLR-independent cell surface delivery but otherwise normal hRAMP1 activity. Sequential shortening of the hRAMP1 transmembrane domain resulted in progressively impaired association with the hCRLR and, as a consequence, abolished CGRP receptor function. In conclusion, the intracellular QSKRT sequence adjacent to the transmembrane domain of hRAMP1 provides a signal for intracellular retention. The sequence is unrelated to consensus endoplasmic reticulum retention/retrieval motives and overridden by the presence of the hCRLR. The entire single transmembrane domain of hRAMP1 together with one hydrophilic amino acid residue at its C-terminus is required for the formation of a fully functional CGRP/hRAMP1/hCRLR receptor complex.

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg -Arrow (Arr) and Frizzled 2 (Fz2) -are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. Here, we analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. Our experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by our in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, we identify a 200 bp regulatory element in the arr locus that can account for the Arr gradient, and we show that this is indirectly negatively controlled by Wg activity. Finally, we analyze the role of Frizzled 3 (Fz3) in this system and find that its expression, which is induced by Wg, contributes to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, our results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system.

125 I]hK KCGRP binding and cAMP formation in response to hK KCGRP were similar to those of hCLR/ myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24 þ 7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [125 I]hK KCGRP binding of hCLR/myc-hRAMP1-C72A was 13 þ 3% of hCLR/mychRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A-and -C104A-expressing cells. Maximal cAMP stimulation by hK KCGRP in hCLR/myc-hRAMP1-C40A-and -C72A-expressing cells was 14 þ 1% and 33 þ 2% of that of the hCLR/myc-hRAMP1 with comparable EC 50 . But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not a¡ected by the deletion of Cys 27 or the substitution of Cys 82 by Ala in hRAMP1, but it was impaired by the substitution of Cys 40 , Cys 57 , Cys 72 and Cys 104 by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.