Recognition sequences and structural elements contribute to shedding susceptibility of membrane proteins

Abstract

Although regulated ectodomain shedding affects a large panel of structurally and functionally unrelated proteins, little is known about the mechanisms controlling this process. Despite a lack of sequence similarities around cleavage sites, most proteins are shed in response to the stimulation of protein kinase C by phorbol esters. The signal-transducing receptor subunit gp130 is not a substrate of the regulated shedding machinery. We generated several chimaeric proteins of gp130 and the proteins tumour necrosis factor alpha (TNF-alpha), transforming growth factor alpha (TGF-alpha) and interleukin 6 receptor (IL-6R), which are known to be subject to shedding. By exchanging small peptide sequences of gp130 for cleavage-site peptides of TNF-alpha, TGF-alpha and IL-6R we showed that these short sequences conferred susceptibility to spontaneous and phorbol-ester-induced shedding of gp130. Importantly, these chimaeric gp130 proteins were functional, as shown by the phosphorylation of gp130 and the activation of signal transduction and activators of transcription 3 ('STAT3') on stimulation with cytokine. To investigate minimal requirements for shedding, truncated cleavage-site peptides of IL-6R were inserted into gp130. The resulting chimaeras were susceptible to shedding and showed the same cleavage pattern as observed in the chimaeras containing the complete IL-6R cleavage site. Surprisingly, we could also generate cleavable chimaeras by exchanging the juxtamembrane sequence of gp130 for the corresponding region of leukaemia inhibitory factor ('LIF') receptor, a protein that like gp130 is not subject to regulated or spontaneous shedding. Thus it seems that there is no minimal consensus shedding sequence. We speculate that structural changes allow the access of the protease to a membrane-proximal region, leading to shedding of the protein.

Molecular and Functional Characterization of a Soluble Form of Oncostatin M/Interleukin-31 Shared Receptor

“…Preliminary results show an induction of sOSMR, after a phorbol ester cell contact, that is counteracted by a metalloprotease inhibitor treatment. 4 This suggests that, in addition to the exon splicing mechanism, sOSMR can also be generated by proteolytic cleavage, similarly to that previously reported for the soluble IL-6R (43)(44)(45).…”

“…Soluble IL-6 receptor is generated two different ways, shedding of the external IL-6R portion or alternative splicing, resulting in the secretion of a soluble form of receptor that lacks the transmembrane domain (43)(44)(45). Two large size signaling receptors belonging to the IL-6 family, gp130 and LIFR, have also been described as soluble products (38,46).…”

Non-Canonical Interleukin 23 Receptor Complex Assembly

“…Deletion variants of sIL-12R␤1 were generated by PCR and splicing by overlapping extension PCR. The resulting cDNAs were subcloned into the eukaryotic expression vector p409 (23). cDNA of the Fc-tagged fusion protein p40_D2D3-p19Fc was generated by PCR.…”

Regulated Shedding of Transmembrane Chemokines by the Disintegrin and Metalloproteinase 10 Facilitates Detachment of Adherent Leukocytes

“…Because the intracellular and transmembrane proportion of CX3CL1 accounts for 6 kDa, both CTFs must be generated by extracellular cleavage in the membrane-proximal region of the chemokine. We next removed the membrane-proximal extracellular part containing the potential cleavage sites as well as the subsequent C-terminal proportion of CX3CL1 by fusing the upper N-terminal part of the CX3CL1 ectodomain to the second FN-like domain of the signal transducer molecule gp130, which is not shed from the cell surface (45) (Fig. 5A).…”