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Xarvcf, Xenopus member of the p120 catenin subfamily associating with cadherin juxtamembrane region.
Paulson AF
,
Mooney E
,
Fang X
,
Ji H
,
McCrea PD
.
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The catenin ARVCF is a member of the p120(ctn) subfamily of Armadillo proteins. A number of catenins directly bind cadherin cytoplasmic tails, contributing to the modulation of cell-cell adhesion and motility processes. Some catenins, such as beta-catenin (and likely p120(ctn)), have additional roles within signaling pathways regulating gene transcription. We have isolated the Xenopus homolog of human ARVCF. Utilizing the cadherin membrane proximal region known to bind p120(ctn) and delta-catenin, coimmunoprecipitation experiments demonstrate that Xarvcf, likewise, binds cadherin in this region and that corresponding point mutations within conserved residues abrogate the Xarvcf-cadherin association. Western blot analysis of Xarvcf protein across a series of developmental stages reveals changes in protein mobility, likely due to changes in phosphorylation. Xarvcf is a maternally provided transcript and expressed in the embryo throughout all stages of development. Interestingly, Xarvcf mRNA is differentially spliced to produce several isoforms, one of which is developmentally regulated. In common with the putative post-translational modifications of the Xarvcf protein, the presence of alternative splice isoforms suggests that Xarvcf possesses the capacity to effect developmental functions in a regulatable manner.
Figure 1
Xarvcf sequence. A, deduced amino acid sequence of Xenopus ARVCF (Xarvcf). Sequence inboldface type represents the central Armadillo domain,shaded sequence indicates the original PCR clone, andparentheses indicate sequence resulting from alternative splicing within the transcript. The sequence data is available from GenBank™ under accession number AF287051. B, alignment of Xarvcf N-terminal sequence with human ARVCF-2 and human p120ctn1.
Figure 2
Schematic of Xarvcf protein domains illustrates the two start sites (1,2), alternative splicing within the C terminus of the protein (insert A) and alternative splicing within the Armadillo domain (inserts B andC). The central Armadillo repeat domain is comprised of 10 repeats with spacer sequence following repeats 4, 5, and 6.
Figure 3
Northern blot analyis of Xarvcf mRNA expression in the Xenopus oocyte. Xarvcf transcripts migrate as a diffuse collection of bands at approximately 6 kb. Each lane contains 5 μg of total RNA isolated from different sets of oocytes.
Figure 4
RT-PCR analysis of Xarvcf expression in earlyXenopus development. A: i, semiquantitative RT-PCR evaluation of total Xarvcf expression at the indicated developmental stages. ii, C-terminal RT-PCR to evaluate the presence of Xarvcf insert A (upper band) during early developmental stages. Without insert A, the PCR band is 216 bp, and, when insert A is present, a PCR product of 333 bp is observed.iii, control RT-PCR reaction of ornithine decarboxylase was used as a loading control. B, Xarvcf schematic to illustrate location of PCR primers within the Xarvcf sequence.
Figure 5
Xarvcf insert A and insert C are specifically expressed in braintissue. Semiquantitative RT-PCR analysis of Xarvcf expression and isoform usage in adult tissues; brain, adipose, heart, stomach, liver, and skin. A: i, RT-PCR of total Xarvcf expression; ii, RT-PCR to detect the C-terminal insert A; iii, RT-PCR to detect the Armadillo domain insert C; iv, control RT-PCR reaction of EF1α. B, Xarvcf schematic to illustrate the location of PCR primers within the Xarvcf sequence.
Figure 6
RT-PCR analysis of alternative splicing at the amino terminus of the Xarvcf transcript. Start site 1 is preferred over start site 2 as made evident via RT-PCR. When the insert is present, a product of 570 bp is generated, and, when absent, a PCR product of 492 bp is produced. Removal of the insert eliminates stop codons residing between start site 1 and the ORF, thereby permitting use of start site 1.
Figure 7
Immunoblot analysis of Xarvcf protein at the indicated embryonic stages. Xarvcf protein in total embryo extracts isolated in the presence of phosphatase inhibitors was detected using affinity-purified anti-Xarvcf peptide polyclonal antibody. The anti-Xarvcf antibody recognizes two major bands at about 100 kDa, the upper band being the most prominent. There is a shift in gel mobility of Xarvcf protein in early development, increasing during cleavage stages (stage 5) and decreasing during gastrulation (stages 10–12.5), likely reflecting developmentally regulated post-translational modification to Xarvcf.
Figure 8
Xarvcf binds to the juxtamembrane region of C-cadherin. A, embryos were injected with mRNA coding for Xarvcf-HA (isoform 1B was used for all of these experiments with hemagglutinin epitope tag) and C-cadherin-MT (myc epitope tag) and were immunoblotted for C-cadherin-MT and Xarvcf-HA following immunoprecipitation of Xarvcf-HA. B, embryos were injected with mRNA coding for Xarvcf-HA and either the membrane targeted C-cadherin JMR-Myr.MT or the mutant C-cadherin JMRGA-Myr.MT. Following immunoprecipitation of C-cadherin JMR-Myr.MT or C-cadherin JMRGA-Myr.MT, the coimmunoprecipitation of Xarvcf-HA protein was detected from Xenopus whole embryo lysates blotted with anti-HA monoclonal antibody. Xarvcf coimmunoprecipitates with membrane-targeted C-cadherin JMR-Myr.MT but not with the mutant construct JMRGA-Myr.MT. C, embryos were injected with mRNA coding for Xarvcf-HA and either the membrane-targeted C-cadherin JMR-Myr.MT or the non-membrane-targeted C-cadherin JMR-MT, lacking the myristylation motif. Immunoprecipitation of JMR-Myr.MT and JMR-MT followed by immunoblotting for Xarvcf-HA showed that the interaction of Xarvcf is stronger with the membrane-localized construct. D, the sequence of the juxtamembrane domain of C-cadherin used to test the interaction of Xarvcf with C-cadherin. Theboxed glycine residues were mutated to alanines for the construct JMRGA-Myr.MT.
