Functional antagonists of sonic hedgehog reveal the importance of the N terminus for activity.
During development, sonic hedgehog functions as a morphogen in both a short-range contact-dependent and in a long-range diffusable mode. Here, we show using a panel of sonic hedgehog variants that regions near the N terminus of the protein play a critical role in modulating these functions. In the hedgehog responsive cell line C3H10T1/2, we discovered that not only were some N-terminally truncated variants inactive at eliciting a hedgehog-dependent response, but they competed with the wild-type protein for function and therefore served as functional antagonists. These variants were indistinguishable from wild-type sonic hedgehog in their ability to bind the receptor patched-1, but failed to induce the hedgehog-responsive markers, Gli-1 and Ptc-1, and failed to promote hedgehog-dependent differentiation of the cell line. They also failed to support the adhesion of C3H10T1/2 cells to hedgehog-coated plates under conditions where wild-type sonic hedgehog supported binding. Structure-activity data indicated that the N-terminal cysteine plays a key regulatory role in modulating hedgehog activity. The ability to dissect patched-1 binding from signaling events in C3H10T1/2 cells suggests the presence of unidentified factors that contribute to hedgehog responses.
Williams
K P
KP
Biogen, Inc., Cambridge, Massachusetts 02142, USA. kevinvwilliams@biogen.com
Rayhorn
P
P
Chi-Rosso
G
G
Garber
E A
EA
Strauch
K L
KL
Horan
G S
GS
Reilly
J O
JO
Baker
D P
DP
Taylor
F R
FR
Koteliansky
V
V
Pepinsky
R B
RB
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D002642:Chick Embryo; D003001:Cloning, Molecular; D004627:Embryonic Induction; D004790:Enzyme Induction; D004926:Escherichia coli; D053823:Hedgehog Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051379:Mice; D008822:Mice, Transgenic; D009420:Nervous System / Q000166:cytology / Q000196:embryology; D015513:Oncogene Proteins / Q000378:metabolism; D009924:Organ Culture Techniques; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010843:Pichia; D011506:Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators; D001616:beta-Galactosidase / Q000235:genetics
D053823:Hedgehog Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D015513:Oncogene Proteins; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010446:Peptide Fragments; D011506:Proteins; C000606240:Ptch1 protein, mouse; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C509239:SHH protein, human; D015534:Trans-Activators; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase