F104S c-Mpl responds to a transmembrane domain-binding thrombopoietin receptor agonist: proof of concept that selected receptor mutations in congenital amegakaryocytic thrombocytopenia can be stimulated with alternative thrombopoietic agents.
OBJECTIVE
To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists.


METHODS
We created cell line models of type II c-Mpl mutations identified in congenital amegakaryocytic thrombocytopenia. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing wild-type or F104S c-Mpl in response to thrombopoietin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD-4665) domains of the receptor by 1-methyltetrazole-5-thiol assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species.


RESULTS
Cells expressing F104S c-Mpl proliferated in response to LGD-4665, but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD-4665 stimulates signaling with delayed kinetics in both wild-type and F104S c-Mpl-expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen-bonding interactions that are critical for binding to thrombopoietin.


CONCLUSIONS
These findings suggest that a small subset of patients with congenital amegakaryocytic thrombocytopenia might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin.
Fox
Norma E
NE
Pediatric Hematology-Oncology, University of California, San Diego, La Jolla, Calif. 92017, USA.
Lim
Jihyang
J
Chen
Rose
R
Geddis
Amy E
AE
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007377:Interleukin-3 / Q000494:pharmacology; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D053628:Receptors, Thrombopoietin / Q000819:agonists* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D013921:Thrombocytopenia / Q000151:congenital / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D034061:Thrombopoiesis / Q000187:drug effects*; D013926:Thrombopoietin / Q000378:metabolism
D007377:Interleukin-3; D010455:Peptides; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C082218:MPL protein, human; D013926:Thrombopoietin