In:
Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 2909-2909
Abstract:
Compounds in the diazo/hydrazino acid class require expression of Tpo receptor (TpoR) for activity. These compounds have demonstrated a remarkable species specificity in their Tpo receptor agonist activity, i.e., the activation of signalling pathways by this class of compounds has only been demonstrated in human and chimpanzee platelets. Platelets of other species demonstrate signalling in response to rhTpo, however, no signalling in response to SB-497115 is detectable by electrophoretic mobility shift assays using platelets of cynomolgus macaques, cat, mouse, mouse, rag, pig, ferret or tree shrew. Additionally, HepG2 cells transiently transfected with human TpoR, but not murine or cynomolgus monkey receptor results in STAT activated-reporter gene activity. To elucidate the mechanism by which this occurs, HepG2 cells were transiently transfected with a STAT-activated reporter gene and various chimeric and mutated receptors and treated with SKF-57626, a tool compound in this diazo class. A series of cyno and human TpoR chimeric receptors were constructed in which the complement receptor homology region 1 (CRH1), CRH2 and the transmembrane (TM) and cytoplasmic domains were interchanged. The functionality of all the chimeric receptors was confirmed by response to rhTpo. The minimal human composition of the chimeric TpoR activated by SKF-57626 was composed of human sequence within the membrane proximal region of the CRH2 domain and the TM domain. Two amino acids in this region are different between cyno and human, a Thr to Ala change in the extracellular CRH2 domain and a Leu at residue 499 in cynomolgus monkeys rather than His in humans in the TM domain. Sequencing of TpoR transmembrane domains of mice, dogs and ferrets, species in which these compounds are inactive, confirmed that the receptor contains Leu499, similar to cynomolgus macaques, while chimpanzee TpoR is similar to human with His at residue 499. To verify the requirement for His499 in the TM domain, a point mutation replacing only Leu499 with His in the cyno TpoR conferred activity when treated with compound. Replacement of His499 with Leu in the human TM domain resulted in an inactive human TpoR in response to compound, but not rhTpo. Further experiments involved mutations in the murine G-CSF receptor (mGCSFR). Human TpoR and mGCSFR have little homology in their TM domains and there is no detectable signalling, proliferation or differentiation responses following treatment of GCSFR expressing cells with either compound or rhTpo. A mGSFR point mutation was tested that contained a His residue nine amino acids into the hydrophobic TM domain of GCSFR, corresponding to His499 in the human and chimpanzee TpoR. HepG2 cells transiently transfected with this receptor and the reporter gene construct responded to compound. A double mutation in which an additional residue three amino acids N-terminal to the His was replaced with Thr, as in the hTpoR exhibited an increase in activity over the single mutation. These results suggest a model in which these TpoR agonist compounds interact with His499, in addition to Thr496, to either change conformation of TpoR or induce dimerization, resulting in activation of the signal transduction pathways of TpoR and imparting biologically relevant function.
Type of Medium:
Online Resource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood.V104.11.2909.2909
Language:
English
Publisher:
American Society of Hematology
Publication Date:
2004
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
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