Localization of disulfide bonds in the frizzled module of Ror1 receptor tyrosine kinase

The frizzled (FRZ) module is a novel module type that was first identified in G-protein-coupled receptors of the frizzled and smoothened families and has since been shown to be present in several secreted frizzled-related pro teins, in some modular proteases, in collagen XVIII, and in various recepto r tyrosine kinases of the Bor family, The FRZ modules constitute the extrac ellular ligand-binding region of frizzled receptors and are known to mediat e signals of WNT family members through these receptors, With an eye toward defining the structure of this important module family, we have expressed the FRZ domain of rat Ror1 receptor tyrosine kinase in Pichia pastoris, By proteolytic digestion and amino acid sequencing the disulfide bonds were fo und to connect the 10 conserved cysteines in a 1-5, 2-4, 3-8, 6-10, and 7-9 pattern. Circular dichroism and differential scanning calorimetry studies on the recombinant protein indicate that the disulfide-bonded FRZ module co rresponds to a single, compact, and remarkably stable folding domain posses sing both alpha -helices and beta -strands.