Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2
Petra Seemann, Raphaela Schwappacher, Klaus W. Kjaer, Deborah Krakow, Katarina Lehmann, Katherine Dawson, Sigmar Stricker, Jens Pohl, Frank Plöger, Eike Staub, Joachim Nickel, Walter Sebald, Petra Knaus, Stefan Mundlos
Petra Seemann, Raphaela Schwappacher, Klaus W. Kjaer, Deborah Krakow, Katarina Lehmann, Katherine Dawson, Sigmar Stricker, Jens Pohl, Frank Plöger, Eike Staub, Joachim Nickel, Walter Sebald, Petra Knaus, Stefan Mundlos
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Research Article Genetics

Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2

Abstract

Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b (BMPR1B) and the BMP antagonist NOGGIN, respectively. We expressed the mutant proteins in limb bud micromass culture and treated ATDC5 and C2C12 cells with recombinant GDF5. Our results indicated that the L441P mutant is almost inactive. The R438L mutant, in contrast, showed increased biological activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like properties. The presented experiments have identified some of the main determinants of GDF5 receptor-binding specificity in vivo and open new prospects for generating antagonists and superagonists of GDF5.

Authors

Petra Seemann, Raphaela Schwappacher, Klaus W. Kjaer, Deborah Krakow, Katarina Lehmann, Katherine Dawson, Sigmar Stricker, Jens Pohl, Frank Plöger, Eike Staub, Joachim Nickel, Walter Sebald, Petra Knaus, Stefan Mundlos

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Expression analysis during joint development and overexpression of Gdf5 ...
Expression analysis during joint development and overexpression of Gdf5 in vivo. (A) In situ hybridization on mouse limb sections at E13.5 and E14.5 with probes specific for Gdf5, Bmp2, Nog, Bmpr1a, and Bmpr1b. The area of joint formation is indicated by arrows. Note strong expression of Gdf5 at both stages but Bmp2 expression in the joint only at E14.5. The longitudinal stripes of Bmp2 expression in the joint area at E13.5 correspond to expression in developing ligaments and not the joint interzone. Nog was expressed in a small band of cells in the joint interzone at E13.5 but not at E14.5. Bmpr1b was expressed in chondrocytes flanking the joint interzone. Bmpr1a shows ubiquitous expression with higher levels in the perichondrium and the developing joints. (B) Overexpression of WT Gdf5 as well as the Gdf5 mutants R438L and L441P in chick embryos using RCAS retroviral system. Alcian blue staining was used to visualize cartilage. For comparison, the injected left limb is shown next to the uninfected right limb, which serves as the control. Note enlargement of skeletal elements, joint fusions, and fusions between digits in WT Gdf5 and R438L mutant infected limbs at stage HH32. Changes in the L441P-injected limb were less severe and were only observed at a later stage (HH34–35). A higher magnification (arrows in B) shows joint fusions. Magnification, ×10 objective (A); ×1.6 objective (WT Gdf5 and R438L in B); ×1.25 objective (L441P in B) (AxioCam HRc camera; Zeiss).