The membrane‐tethered mucins are cell surface‐associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane‐associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane‐tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site‐directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately recreate an accessible cleavage site.
AS (Apert syndrome) is a congenital disease composed of skeletal, visceral and neural abnormalities, caused by dominant-acting mutations in FGFR2 [FGF (fibroblast growth factor) receptor 2]. Multiple FGFR2 splice variants are generated through alternative splicing, including PTC (premature termination codon)-containing transcripts that are normally eliminated via the NMD (nonsense-mediated decay) pathway. We have discovered that a soluble truncated FGFR2 molecule encoded by a PTC-containing transcript is up-regulated and persists in tissues of an AS mouse model. We have termed this IIIa-TM as it arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 [TM (transmembrane domain)]. IIIa-TM is glycosylated and can modulate the binding of FGF1 to FGFR2 molecules in BIAcore-binding assays. We also show that IIIa-TM can negatively regulate FGF signalling in vitro and in vivo. AS phenotypes are thought to result from gain-of-FGFR2 signalling, but our findings suggest that IIIa-TM can contribute to these through a loss-of-FGFR2 function mechanism. Moreover, our findings raise the interesting possibility that FGFR2 signalling may be a regulator of the NMD pathway.
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