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Mol Cell Biol, June 1998, p. 3321-3329, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mutations in the Extracellular Domain Cause RET
Loss of Function by a Dominant Negative Mechanism
Maria Pia
Cosma,1
Monica
Cardone,1
Francesca
Carlomagno,2 and
Vittorio
Colantuoni1,3,*
Dipartimento di Biochimica e Biotecnologie
Mediche and Centro di Ingegneria Genetica,
CEINGE,1 and
Centro di Endocrinologia e
Oncologia Sperimentale del CNR, Dipartimento di Biologia e Patologia
Cellulare e Molecolare, Facoltà di Medicina e
Chirurgia, Università di Napoli "Federico
II",2 Naples, and
Facoltà di
Farmacia, Università di Reggio Calabria,
Catanzaro,3 Italy
Received 26 February 1998/Accepted 19 March 1998
The RET proto-oncogene encodes a tyrosine kinase
receptor expressed in neuroectoderm-derived cells. Mutations in
specific regions of the gene are responsible for the tumor syndromes
multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B), while
mutations along the entire gene are involved in a developmental
disorder of the gastrointestinal tract, Hirschsprung's disease (HSCR
disease). Two mutants in the extracellular domain of RET, one
associated with HSCR disease and one carrying a flag epitope, were
analyzed to investigate the impact of the mutations on RET function.
Both mutants were impeded in their maturation, resulting in the lack of
the 170-kDa mature form and the accumulation of the 150-kDa immature
form in the endoplasmic reticulum. Although not exposed on the cell
surface, the 150-kDa species formed dimers and aggregates; this was
more pronounced in a double mutant bearing a MEN 2A mutation. Tyrosine
phosphorylation and the transactivation potential were drastically
reduced in single and double mutants. Finally, in cotransfection
experiments both mutants exerted a dominant negative effect over
protoRET and RET2A through the formation of a
heteromeric complex that prevents their maturation and function. These
results suggest that HSCR mutations in the extracellular region cause RET loss of function through a dominant negative mechanism.
*
Corresponding author. Mailing address: Dipartimento di
Biochimica e Biotecnologie Mediche, Facoltà di Medicina e
Chirurgia, Università di Napoli, "Federico II," Via Sergio
Pansini, 5, 80131 Naples I, Italy. Phone: 39 81 746 3735. Fax: 39 81 746 3074. E-mail: colantuoni{at}dbbm.unina.it.
Mol Cell Biol, June 1998, p. 3321-3329, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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