AKR-501 Activates the Thrombopoietin Receptor through Interaction with the Transmembrane Domain

AKR-501 (1-(3-Chloro-5-{[4-(4-chloro-2-thienyl)-5-(4-cyclohexylpiperazin-1-yl) thiazol-2-yl]carbamoyl}-2-pyridyl)piperidine-4-carboxylic acid, formerly known as YM-477) is an orally-active full agonist of the thrombopoietin (TPO) receptor, c-Mpl. Human c-Mpl consists of an N-terminal extracellular domain containing a membrane-distal cytokine receptor homology region (CRH-1) which is the site of TPO binding, as well as a membrane-proximal CRH region (CRH-2) with yet to be identified functions. The C-terminal end of c-Mpl contains the transmembrane domain (TM), and a cytoplasmic signal transduction domain. It has previously been demonstrated that AKR-501 does not interfere with TPO binding to c-Mpl at the CRH-1 domain, and that AKR-501 is highly species-specific with action only with human and chimpanzee c-Mpl. In the current study, we sought to define the AKR-501 binding domain on c-Mpl by constructing a series of human and mouse chimera c-Mpl receptors in order to evaluate which domain(s) of human c-Mpl confers growth stimulation in response to AKR-501. We found that Ba/F3 cells expressing a chimeric c-Mpl that contained both human CRH-2 and TM responded to AKR-501 and stimulated growth to the same extent as TPO. Other chimeras with only human CRH-2 could not, suggesting that TM is critical for AKR-501 action. A construct was then built to substitute the mouse TM leucine amino acid residue at position 499 with the corresponding human histidine amino acid; this residue is conserved between humans and chimpanzee, but is not found in other species. We found the mouse c-Mpl with a His499 substitution was sufficient to support AKR-501 stimulated growth of Ba/F3 cells. Thus, we concluded that the c-Mpl TM appears essential for AKR-501 activity, and the species specific activity of AKR-501 depends on His499 in the TM.