The amyloid precursor protein (APP) is a single-span integral membrane protein whose C-terminal fragment C99 is cleaved within the transmembrane helix by γ-secretase. Cleavage produces various Aβ peptides that are linked to the etiology of Alzheimer's disease. The transmembrane helix is known to homodimerize in a sequence-specific manner, and considerable controversy about whether the homodimeric form of C99 is cleaved by γ-secretase exists. Here, we generated various covalent C99 homodimers via cross-linking at engineered cysteine residues. None of the homodimers was cleaved in vitro by purified γ-secretase, strongly suggesting that homodimerization protects C99 from cleavage.

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Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.