Functional characterization of c-Mpl ectodomain mutations that underlie congenital amegakaryocytic thrombocytopenia.
Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F'-G' loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT.
The Walter and Eliza Hall Institute of Medical Research , Parkville, Victoria , Australia and.
Leila N
LN
Varghese
Jian-Guo
JG
Zhang
Samuel N
SN
Young
Tracy A
TA
Willson
Warren S
WS
Alexander
Nicos A
NA
Nicola
Jeffrey J
JJ
Babon
James M
JM
Murphy
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D000080984:Congenital Bone Marrow Failure Syndromes; D008533:Megakaryocytes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D015513:Oncogene Proteins / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011981:Receptors, Prolactin / Q000235:genetics; D053628:Receptors, Thrombopoietin / Q000235:genetics*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000235:genetics; D013921:Thrombocytopenia / Q000235:genetics*; D013926:Thrombopoietin / Q000378:metabolism*
C481270:Mpl protein, mouse; D015513:Oncogene Proteins; D011981:Receptors, Prolactin; D053628:Receptors, Thrombopoietin; D013926:Thrombopoietin