Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding

Abstract

Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Since shedding rapidly and irreversibly alters the characteristics of cells, this process is strictly regulated. However, the molecular mechanisms determining the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding-susceptibility of membrane proteins.

Strategies to Target ADAM17 in Disease: From Its Discovery to the iRhom Revolution

“…Such an analysis would be useful to uncover molecular determinants that render shedding of ADAM17 substrates as iRhom1- or iRhom2-dependent. Finally, negatively charged residues within the stalk region of ADAM17 substrates emerged as pivotal in directing their shedding, as they confer shedding resistance to a number of ADAM17 substrates [ 81 ]. Although not proven yet, given the crucial role of iRhoms in ADAM17 activity, these results suggest that iRhoms, additionally to ADAM17, may take contacts with its substrates, which can be disrupted by negatively charged residues within their stalk region.…”

ALCAM: A Novel Surface Marker on EpCAMlow Circulating Tumor Cells

“…There are two known isoforms, “short” and “long”, differing in only a stretch of 12 negatively charged amino acids located in the extracellular domain. The two variants are produced by alternate splicing and are differentially susceptible to cleavage by ADAM metallopeptidase domain 17 or 10 (ADAM17/10) [ 82 , 83 ]. It was expressed in nearly 100% of the cell population among almost all of the three CTC lines that were analyzed ( Figure 4 i).…”

To cut or not to cut: New rules for proteolytic shedding of membrane proteins

“…The introduction of negative charges into the juxtamembrane may happen under physiological conditions upon alternative splicing of juxtamembrane domain-encoding exons. Iwagishi et al (4) show this to be the case for ALCAM. The alternatively spliced exon encodes for 14 amino acids, six of which are aspartate and glutamate in the nonshed isoform, whereas there is only a single glutamate in the efficiently shed isoform.…”