Differential responsiveness of tumour necrosis factor receptors (TNFR) type 1 and 2 : the critical role of the TNFR stalk region

Abstract

The pro-inflammatory cytokine tumour necrosis factor (TNF) exerts its bioactivity via two plasma membrane receptors, TNF receptor (TNFR)1 and TNFR2. Both receptors are fully activated by membrane-bound TNF, while soluble TNF (sTNF) activates only TNFR1 efficiently. Preliminary data from our group suggest that the membrane proximal extracellular region (stalk region) and/or the transmembrane region of the TNFR control the differential responsiveness to sTNF. The aims of this project were to ascertain the region determining sTNF responsiveness and to investigate the underlying molecular mechanism(s). Fibroblasts from tnfr1-/-/tnfr2-/- double knockout mice were stably transfected with chimaeras consisting of the extracellular and transmembrane domain of TNFR and the intracellular portion of Fas (TNFR-Fas). Using this cellular system, I could show that 42 amino acids within the TNFR2 stalk region control responsiveness to sTNF. Replacement of the stalk regions of TNFR1-Fas and TNFR2-Fas with artificial linkers proved that stalk region length does not determine differential responsiveness. Furthermore, responsiveness to sTNF was not affected when either conserved proline residues or O-glycosylation sites in the TNFR2 stalk region were mutated. Moreover, partial replacement of the TNFR2 stalk region with overlapping artificial linkers also left sTNF responsiveness unaltered. Therefore, the underlying molecular mechanism controlling responsiveness to sTNF appears to be more complex and remains to be elucidated. Importantly, the critical role of the TNFR stalk region in sTNF responsiveness could also be confirmed for wild type TNFR2 at the level of signalling complex formation. Data from chemical crosslinking and confocal microscopy studies revealed that the stalk region controls ligand-independent receptor pre-assembly and formation of larger receptor clusters. Taken together, data obtained in this PhD thesis indicate that the TNFR2 stalk region is a major determinant of differential sTNF responsiveness and ligand-independent receptor-receptor interactions, the latter being a potential prerequisite for the formation of larger ligand/receptor clusters and signal initiation.