NMR Structural Studies of Interactions of a Small, Nonpeptidyl Tpo Mimic with the Thrombopoietin Receptor Extracellular Juxtamembrane and Transmembrane Domains

Thomas H Marsilje,Shin-Shay Tian,H Martin Seidel,Stanley J Opella,Min-Ju Kim,Sang Ho Park,Pierre-Yves Michellys

doi:10.1074/jbc.m611616200

Abstract

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.

Highlights

Changes in gene regulation, which are thought to promote progression of the Tpo receptor (TpoR)-expression cells along the megakaryocytic pathway
These results suggested that the human TpoR (hTpoR) TM domain is either involved in interaction with SB394725 or is required for productive conformational changes induced by binding of SB394725 to hTpoR
To understand the agonist activity of SB394725, we studied its interaction with the extracellular juxtamembrane region (JMR)-TM domain of hTpoR by solution and solid-state NMR using a 41-residue hTpoR-(479 –519) polypeptide, which consists of 13 extracellular JMR residues, 22 predicted TM domain residues, and 6 cytoplasmic JMR residues (Fig. 2A)

Summary

Introduction

Changes in gene regulation, which are thought to promote progression of the TpoR-expression cells along the megakaryocytic pathway. These data suggested that, in addition to the TM domain residues, the 13 amino acids in the extracellular JMR of hTpoR are likely to be involved with or influenced by interaction of the receptor with SB394725. Our results from the domain swap and mutagenesis studies suggested that the extracellular JMR-TM region of hTpoR plays a key role in interacting with SB394725.

Results

Conclusion