Loss of KDEL function from a calreticulin frameshift mutation drives expression of an immature, mutant calreticulin-dependent form of the thrombopoietin receptor MPL

Abstract

Mutant calreticulin (CALR) activates the thrombopoietin (TPO) receptor MPL, thereby inducing the development of essential thrombocythemia and primary myelofibrosis. Mutant CALR, due to a frameshift mutation, loses the endoplasmic reticulum (ER) retention signal, the KDEL sequence and is released extracellularly. To examine the significance of the loss of the KDEL sequence in mutant CALR-induced MPL activation, a series of constructs were prepared, including mutant CALR plus KDEL (mutant CALRKDEL), mutant CALR plus the Venus tag and KDEL (mutant CALRVenus-KDEL), and wild-type CALR minus KDEL (CALR WTΔKDEL). UT-7/TPO cells expressing mutant CALRKDEL exhibited autonomous cell growth in the absence of TPO, accompanied by the extracellular secretion of mutant CALRKDEL and subsequent activation of MPL. In contrast, UT-7/TPO cells expressing mutant CALRVenus-KDEL did not exhibit autonomous cell growth or MPL activation without TPO as well as the reduced extracellular secretion of mutant CALRVenus-KDEL. These results suggest that the loss of KDEL function in mutant CALR is closely linked to MPL activation and the extracellular secretion of mutant CALR. While UT-7/TPO cells expressing CALR WTΔKDEL did not exhibit autonomous cell growth, they were responsive to mutant CALR proteins added exogenously, as evidenced by STAT5 activity. Furthermore, CALR WTΔKDEL conferred mutant CALR sensitivity to MPL by recognizing the N-glycans of MPL while maintaining it in an immature form, which may bind to mutant CALR. In conclusion, deletion of the ER retention signal KDEL from CALR is a prerequisite for the expression of the immature form of MPL, which can interact with secreted mutant CALR.