Transmembrane Domain Structure and Function in the Erythropoietin Receptor
Brett, Ian Christopher
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Activation of the erythropoietin receptor (EpoR) by the soluble cytokine erythropoietin (Epo) is essential for the differentiation of erythrocyte progenitors and their development into red blood cells. The single transmembrane (TM) helix of the EpoR mediates dimerization of the receptor in the inactive state and is responsible for coupling ligand binding to activation of an intracellular Janus kinase. Neither the structure of the inactive dimer nor the structural changes in the TM region that occur upon ligand binding are known. This work presents the solution NMR structures of peptides corresponding to the TM and juxtamembrane (JM) sequences that bridge the extracellular and intracellular domains. The N-terminal end of the TM-JM peptides contains the transition point between the last &beta -strand of the extracellular D2 domain of the receptor and the TM &alpha -helix. NMR measurements indicate that the TM helix extends to Pro225. This proline allows Asp224 to fold back and form side chain hydrogen bonds to the backbone NH of Leu226. Structural studies on the TM region of the EpoR alone reveal intermolecular contacts between polar residues (Ser231, Ser238 and Thr242). At the intracellular TM-JM boundary, the defined &alpha -helical structure appears to break at Arg250-Arg251. However, Leu253-Lys256 exhibit downfield carbonyl chemical shifts consistent with helical structure for the JM switch region. To stabilize the TM-JM peptides in an active conformation, two approaches were undertaken. The first approach was to substitute Leu223 with cysteine full length L223C EpoR is constitutively active. The second approach was to characterize the complex between the TM-JM dimer and the TM domain of an EpoR-activating viral membrane protein, gp55-P. In both cases, the largest chemical shift changes were at the intracellular TM-JM boundary, particularly His249. Mechanisms of receptor activation that unite biophysical and biochemical data are discussed.