Dataset Information

Cx32 wt vs mutant purified proteins

ABSTRACT: An analysis of purified Cx32 wt, W3S and R22G mutations. The three different proteins were expressed, digested and measured in DIA.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Dina Schuster

LAB HEAD: Volodymyr Korkhov

PROVIDER: PXD033848 | Pride | 2023-08-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20220505_112100_220505_Cx32_mutations.sne	Other
	20220505_162141_dschuster_Report.csv	Csv
	20220505_162141_dschuster_Report.log.txt	Txt
	20220505_162141_dschuster_Report.params	Other
	20220505_162141_dschuster_Report.setup.txt	Txt

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Project description:The haematopoietic cytokine thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for maintenance of the haematopoetic stem cell compartment. Tpo is a heavily glycosylated, hepatocyte-derived cytokine which functions by binding to its receptor (TpoR) on target cells and thereby activating intracellular signalling cascades that induce their proliferation and/or differentiation. In addition to its role in signal propagation, TpoR is expressed on the surface of platelets, where it contributes to regulation of Tpo levels by sequestering circulating cytokine. TpoR belongs to the homodimeric Class I cytokine receptor family but is unusual due to a duplication of the Cytokine binding Homology Region (CHR). Almost thirty years after initial discovery of TpoR, the structure of the human Tpo:TpoR interaction was recently reported. Here we determine the structure of extracellular portion of the murine Tpo:TpoR signalling complex using single particle cryo-EM. The structure reveals that Tpo:TpoR forms a largely symmetrical 1:2 complex. The cytokine cross-links the same site on the membrane-distal CHR of both receptor chains using opposing surfaces and with significantly different affinities. This orients the two membrane-proximal CHRs such that they contact one another adjacent to the plasma membrane. The potential cytokine-binding site in CHR2 is glycosylated and does not interact with Tpo. A large insertion in CHR1 that is unique to Tpo forms a partially structured loop that is disulphide bonded to CHR2 and, in one receptor chain, contacts cytokine. Biochemical analyses indicate that the glycosylated C-terminal domain of Tpo does not influence receptor binding. We demonstrate that the therapeutic TpoR agonist Romiplostim binds to the same site on the receptor as does cytokine. Our study characterises the Tpo/TpoR interaction structurally and biochemically to allow for the future development of potent TpoR agonists for therapeutic use.

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Cx32 wt vs mutant purified proteins

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