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

Although regulated ectodomain shedding affects a large panel of structurall y and functionally unrelated proteins, little is known about the mechanisms controlling this process. Despite a lack of sequence similarities around c leavage sites, most proteins are shed in response to the stimulation of pro tein kinase C by phorbol esters. The signal-transducing receptor subunit gp 130 is not a substrate of the regulated shedding machinery. We generated se veral chimaeric proteins of gp130 and the proteins tumour necrosis factor a lpha (TNF-alpha), transforming growth factor a (TGF-alpha) acid interleukin 6 receptor (IL-6R), which are known to be subject to shedding. By exchangi ng small peptide sequences of gp 130 for cleavage-site peptides of TNF-alph a, TGF-alpha and 1L-6R we showed that these short sequences conferred susce ptibility to spontaneous and phorbol-ester-induced shedding of gp130. Impor tantly, these chimaeric gp130 proteins were functional, as shown by the pho sphorylation of gp130 and the activation of signal transduction and activat ors of transcription 3 ('STAT3') on stimulation with cytokine. To investiga te minimal requirements fur shedding, truncated cleavage-site peptides of I L-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 ge nerate cleavable chimaeras by exchanging the juxtamembrane sequence of gp13 0 for the corresponding region of leukaemia inhibitor factor ('LIF') recept or, a protein that like gp130 is not subject to regulated or spontaneous sh edding. 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.