Functional characterization of human NTRK2 mutations identified in patients with severe early-onset obesity.
OBJECTIVE
The neurotrophin receptor TrkB has been implicated in the regulation of energy homeostasis in rodents. We have previously identified four rare missense mutations in the gene encoding TrkB, NTRK2, in 198 severely obese children with developmental delay. We have now undertaken a more detailed analysis of the in vitro functional consequences of the mutations identified: I98V, P660L, T821A and Y722C.


METHODS
Wild-type and mutant TrkB receptor constructs were stably transfected into PC12 cells and the signaling responses to the endogenous ligand, brain-derived neurotrophic factor (BDNF), were examined by Western blotting of cell lysates. In the case of Y722C, PC12 cells stably expressing this mutant were studied for their ability to respond to BDNF by promoting neurite outgrowth and cell survival.


RESULTS
Further functional characterization of the previously reported Y722C TrkB mutation reveals impaired activation of mitogen-activated protein kinase, phospholipase C-gamma and Akt, as well as reduced BDNF-induced neurite outgrowth and cell survival in stably transfected PC12 cell lines. However, the signaling properties of I98V, P660L and T821A were all indistinguishable from wild type.


CONCLUSIONS
We provide further evidence for the impairment in signaling by Y722C and show that as well as a loss of signaling, this mutation affects the ability of TrkB to promote neurite outgrowth in response to BDNF. Thus, impaired hypothalamic neurogenesis may contribute to the severe hyperphagia and obesity seen in the individual harboring the Y722C variant. The other three rare TrkB variants do not show reduced autophosphorylation or impaired downstream signaling in vitro and, as yet, it is unclear whether these variants contribute to obesity in these patients.
Gray
J
J
Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.
Yeo
G
G
Hung
C
C
Keogh
J
J
Clayton
P
P
Banerjee
K
K
McAulay
A
A
O'Rahilly
S
S
Farooqi
I S
IS
D017668:Age of Onset; D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis / Q000502:physiology; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D002648:Child; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D016501:Neurites / Q000502:physiology; D009765:Obesity / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology; D010766:Phosphorylation; D051381:Rats; D020813:Receptor, trkB / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction; D014162:Transfection
D019208:Brain-Derived Neurotrophic Factor; D020813:Receptor, trkB