A Structure-Function Analysis Revealed Histidine in the Transmembrane Domain of Human Thrombopoietin Receptor Is Essential for a Novel Non-Peptidyl Thromobopoietin Mimetics, NIP-004, To Induce Signaling for Cellular Proliferation.

NIP-004 is a novel synthetic compound that displays human (Hu) thrombopoietin receptor (Mpl) agonistic activity. NIP-004 stimulated proliferation of HuMpl-expressing cell lines such as UT-7/EPO-HuMpl and Ba/F3-HuMpl, but not murine (Mu) Mpl-expressing cell lines such as UT-7/EPO-MuMpl and Ba/F3-MuMpl. NIP-004 or its derivative compound stimulated colony formation of CD41a⁺ megakaryocytes from human bone marrow (BM)-derived CD34⁺ hematopoietic progenitor cells, but not from murine BM cells or cynomolgus or rhesus monkey BM-derived CD34⁺ cells. These results indicated that NIP-004 has strict species specificity. To identify a molecular basis for the species specificity displayed by NIP-004, we analyzed the amino acid sequence of Mpl from two non-human primates, cynomolgus and rhesus. Cynomolgus Mpl displayed the highest level of sequence homology, 96% identical to HuMpl, with only 22 amino-acids differing between the two species, and 14 residues in the 22 amino-acids of HuMpl differed from rhesus Mpl. Comparison of these 14 amino-acid residues as either hydrophobic or hydrophilic and either electrically charged or uncharged revealed distinguishing features of an amino acid residue. Histidine (His) at position 499 from the N-terminal residue in the transmembrane domain of HuMpl is specific for humans. We thus performed site-directed mutagenesis this residue in HuMpl and MuMpl, and analyzed STAT5 activation via each receptor in the Hek293 human embryonic kidney cell line using STAT-reporter gene assay. Wild-type HuMpl, but not MuMpl, activated STAT5 after stimulation with NIP-004. HuMpl with His⁴⁹⁹ mutated to Leu (HuMpl^H499L) failed to induce STAT5 activation via NIP-004 stimulation. Conversely, when MuMpl was engineered to contain His⁴⁹⁰ (MuMpl^L490H), it was then capable of activating STAT5. Furthermore, NIP-004 stimulated proliferation of Ba/F3-MuMpl^L490H cells, but not Ba/F3-HuMpl^H499L cells. Western blotting analysis revealed that NIP-004 induced phosphorylation of STAT5 proteins in Ba/F3-HuMpl and Ba/F3-MuMpl^L490H cells, but not Ba/F3-MuMpl and Ba/F3-HuMpl^H499L cells. All Ba/F3 transfectants expressing these Mpl constructs responded to TPO stimulation. These observations indicate that His in the transmembrane domain of Mpl is an essential residue for the ability of NIP-004 to act as an Mpl agonist. We also found none of other experimental animals such as common marmoset, squirrel monkey, beagle dog, Japanese White rabbit, Syrian hamster, Hartley guinea pig and Wistar rat with His in the transmembrane of Mpl. Although His exists in the transmembrane domain of human G-CSFR, NIP-004 failed to stimulate the proliferation of Ba/F3 cells expressing human G-CSFR. In conclusion, a novel non-peptidyl synthetic compound, NIP-004, displays Mpl agonistic activity with strict species specificity via His in the transmembrane domain of Mpl.