Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanism.
Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function.
Hime
Gary R
GR
Department of Anatomy and Cell Biology, University of Melbourne, Australia. grhime@unimelb.edu.au
Lada
Hania
H
Fietz
Michael J
MJ
Gillies
Susan
S
Passmore
Abraham
A
Wicking
Carol
C
Wainwright
Brandon J
BJ
D000595:Amino Acid Sequence; D000818:Animals; D001478:Basal Cell Nevus Syndrome / Q000235:genetics; D019521:Body Patterning; D004330:Drosophila / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029721:Drosophila Proteins; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D017434:Protein Structure, Tertiary / Q000235:genetics; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D012469:Salivary Glands / Q000033:anatomy & histology / Q000166:cytology; D000072086:Smoothened Receptor; D014921:Wings, Animal / Q000033:anatomy & histology / Q000166:cytology
D029721:Drosophila Proteins; D008565:Membrane Proteins; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; C100664:smo protein, Drosophila