Cloning and functional characterization of a novel c-mpl variant expressed in human CD34 cells and platelets.
The thrombopoietin receptor, c-mpl, is a crucial element not only in thrombopoietin (TPO)-initiated signaling pathways but also in the regulation of the circulating amount of TPO. We have identified a new c-mpl isoform, called c-mpl-del, that lacks 72 bp (24 amino acids) in the extracellular region of c-mpl and arises as a consequence of alternative RNA splicing between exons 8 and 9. c-mpl-del is expressed along with c-mpl-wt in blood mononuclear cells, CD34(+)cells, megakaryocytes, and platelets prepared from either normal donors or ET patients, although its relative expression appears to increase with megakaryocyte differentiation. The c-mpl-del-transfected cells expressed greater amounts of c-mpl-del RNA and protein than the comparable c-mpl-wt-transfected cells, however flow cytometry analysis could not detect any c-mpl receptor on the surface of the c-mpl-del-transfected cells. Further evidence for the absence of surface c-mpl-del was that in contrast to cells transfected with c-mpl-wt, those transfected with c-mpl-del did not grow in response to TPO, failed to undergo tyrosine phosphorylation of TPO-specific signal molecules, and did not bind(125)I-rHuTPO. Taken together, these results demonstrate that c-mpl-del, a naturally occurring variant of c-mpl, fails to be incorporated into the cell membrane but might serve as a mechanism to decrease the overall expression of functional c-mpl late in megakaryocyte differentiation.
Li
J
J
Hematology/Oncology Unit, Massachusetts General Hospital, Boston 02114, USA.
Sabath
D F
DF
Kuter
D J
DJ
D017398:Alternative Splicing; D018952:Antigens, CD34; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011971:Receptors, Immunologic / Q000235:genetics / Q000502:physiology*; D053628:Receptors, Thrombopoietin; D013926:Thrombopoietin / Q000378:metabolism*; D014162:Transfection
D018952:Antigens, CD34; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D011971:Receptors, Immunologic; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D013926:Thrombopoietin