Hedgehog (Hh) signaling proteins stimulate cell proliferation, differentiation, 
and tissue patterning at multiple points in animal development. A single Hh 
homolog is present in Drosophila, but three Hh homologs, Sonic Hh, Indian Hh, 
and Desert Hh, are present in mammals. Distribution, movement, and reception of 
Hh signals are tightly regulated, and abnormal Hh signaling is associated with 
developmental defects and cancer. In addition to the integral membrane proteins 
Patched and Smoothened, members of the Drosophila Ihog family of adhesion-like 
molecules have recently been shown to bind Hh proteins with micromolar affinity 
and positively regulate Hh signaling. Cell adhesion molecule-related, 
down-regulated by oncogenes (CDO) and Brother of CDO (BOC) are the closest 
mammalian relatives of Drosophila Ihog, and CDO binds Sonic Hh with micromolar 
affinity and positively regulates Hh signaling. Despite these similarities, 
structural and biochemical studies have shown that Ihog and CDO utilize 
nonorthologous domains and completely different binding modes to interact with 
cognate Hh proteins. We report here biochemical and x-ray structural studies of 
Sonic, Indian, and Desert Hh proteins both alone and complexed with active 
domains of CDO and BOC. These results show that all mammalian Hh proteins bind 
CDO and BOC in the same manner. We also show that interactions between Hh 
proteins and CDO are weakened at low pH. Formation of Hh-mediated Hh oligomers 
is thought to be an important feature of normal Hh signaling, but no conserved 
self-interaction between Hh proteins is apparent from inspection of 14 
independent Hh-containing crystal lattices.