ES cells were electroporated with 10 g of SstI linearized CRFB4 targeting vector per 107 cells and grown under double selection in 400 g/ml G-418 and 1 M gancyclovir (Roche Labs., Nutley, NJ). sequence and structural resemblance, notably evolutionary links to fibronectin type III within domains of the extracellular receptor portion of 100 amino acids (1). The family includes tissue factor (TF; 2, 3), a ligand-binding subunit of the IL-10 receptor (IL-10R1; 4, 5), two subunits of the IFN- receptor (IFN-R1 and IFN-R2; 6C8), two subunits of the IFN- receptor (IFN-R1 Rabbit Polyclonal to ARF4 and IFNAR2; 9C11), and the orphan receptor CRF2-4 (12, 13). CRF2 members are characterized by the presence of a 200Camino acid extracellular domain composed of two distinct subdomains of 100 amino acids (1). These subdomains contain conserved cysteine, proline, and tryptophan residues that determine a characteristic folding pattern of seven beta strands similar to the constant domain of immunoglobulins (14). Family members also contain conserved intracellular domain regions that are involved in the interaction with downstream signaling molecules (15). Full length CRFB4 (the gene that encodes CRF2-4) cDNA was cloned based on an expressed sequence tag corresponding to the D21S58 locus on human chromosome 21 that maps close to IFNAR1 (12, 16). The genes encoding IFN-R2, CRF2-4, IFN-R1, and IFN-R2 form a cluster on human chromosome 21 (11). Human CRFB4 cDNA was also independently cloned through exon trapping and used as Canertinib dihydrochloride a probe to isolate a murine homologue that encodes a 349Camino acid polypeptide that is 69% identical to the 325-residue human counterpart (13). CRFB4 maps on chromosome 16 to a region of conserved synteny with human chromosome 21 (16). This clustering and the similarity to IFN receptor genes (17) suggested that CRF2-4 may be a component of IFN receptors, but coexpression of human CRF2-4 with human IFN-R1 and/or human IFN-R2 in a mouse L929 cell line did not affect the responsiveness of such cells to human IFNs (Gibbs, V.C., unpublished data). Whereas functional type I and type II IFN receptors have been reconstituted from their known subunits (14, 18), there is evidence to Canertinib dihydrochloride suggest that other CRF2 members may need additional receptor subunits to transduce biological responses. TF is a high affinity receptor for plasma factor VII/VIIa and is responsible for cellular initiation of the coagulation protease cascade (2). There is evidence to suggest that TF that has only a 20Camino acid cytoplasmic domain may participate in intracellular signaling through association with an Canertinib dihydrochloride unknown coreceptor. Thus, inactivation of the TF gene in the mouse resulted in early embryonic lethality, presumably due to anomalies in blood vessel formation (19, 20). There is some biochemical evidence to suggest that, in monocytes, TF may associate with a component of the IgE receptor (21), but TF-mediated signaling pathways Canertinib dihydrochloride remain elusive. IL-10R1 is able to bind IL-10 and signal a biological response, but there is evidence to suggest that a coreceptor is involved. Intriguingly, human IL-10 proved more active on mouse Ba/F3 cells transfected with the murine IL-10R1 than on the same cells expressing the human IL-10R1 (5), suggesting that these murine cells may express a species-specific coreceptor. Characterization of an EBV-derived IL-10 homologous protein (22) provided further evidence for the requirement of additional receptor components for signaling. Notably, a soluble form of human IL-10R1 neutralized biologic responses only to human, but not to EBV-derived, IL-10 (5). Lastly, IL-10 treatment of T cells and monocytes triggers activation of the Jak1 and Tyk2 kinases (23, 24). The known receptor subunit coprecipitated only with Jak1, suggesting that Tyk2 may associate with a second receptor subunit (23, 24). Using hybrid IFN-R2/CRF2-4 molecules, it has recently been shown that CRF2-4 associates with Tyk2 (25), and.