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 proteins, in some modular proteases, in collagen XVIII, and in various receptor tyrosine kinases of the Ror family. The FRZ modules constitute the extracellular ligand-binding region of frizzled receptors and are known to mediate 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 found 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 corresponds to a single, compact, and remarkably stable folding domain possessing both ␣-helices and -strands.Genes of the frizzled receptor family encode seven-transmembrane proteins that act as receptors for secreted Wnt glycoproteins (1). The extracellular regions of these receptors consist of about 120 residues containing 10 highly conserved cysteines. The extracellular cysteine-rich domain has been shown to be necessary and sufficient for Wnt ligand binding by frizzled receptors (2). Recently, several groups have shown independently that related domains (hereafter called FRZ 1 modules for frizzled-related modules) are found in diverse modular proteins from Caenorhabditis elegans and Drosophila melanogaster to vertebrates (3-6). In addition to frizzled receptors and the related seven-transmembrane smoothened receptors, these modular proteins include secreted frizzled-related proteins, carboxypeptidase Z, collagen ␣1 XVIII, the serine protease lipoprotein receptor-related protein 4 (7), and several members of the Ror subfamily of receptor tyrosine kinases (Fig. 1).Different members of the Ror family fulfill diverse biological functions. The muscle-specific tyrosine kinase (MuSK) has been shown to be indispensable for the formation of the neuromuscular junction because it is part of a receptor complex that mediates the action of agrin (8 -10). The Drosophila proteins Dror and Dnrk have been implicated in the development of the nervous system because during embryonic development expression of these proteins is restricted to neuronal tissues (11,12). A more general role is suggested for the C. elegans Ror homologue, cam-1, which has been shown to guide migrating cells and orient the polarity of asymmetric cell divisions and axon outgrowth (13).Recent studies suggest that vertebrate Ror1s and Ror2s may have distinct biological roles. Oishi et al. (14) have shown that during embryogenesis, expression of Rorl is sustained in the nervous system and is also detected in non-neuronal tissues after birth. In contrast, the expression of Ror2 declines after birth. ...