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Growth factor and co-receptor release by structural regulation of substrate metalloprotease accessibility

View Article: PubMed Central - PubMed

ABSTRACT

Release of cytokines, growth factors and other life-essential molecules from precursors by a-disintegrin-and-metalloproteases (ADAMs) is regulated with high substrate-specificity. We hypothesized that this is achieved by cleavage-regulatory intracellular-domain (ICD)-modifications of the precursors. We show here that cleavage-stimuli-induced specific ICD-modifications cause structural substrate changes that enhance ectodomain sensitivity of neuregulin-1 (NRG1; epidermal-growth-factor) or CD44 (receptor-tyrosine-kinase (RTK) co-receptor) to chymotrypsin/trypsin or soluble ADAM. This inside-out signal transfer required substrate homodimerization and was prevented by cleavage-inhibitory ICD-mutations. In chimeras, regulation could be conferred to a foreign ectodomain, suggesting a common higher-order structure. We predict that substrate-specific protease-accessibility-regulation controls release of numerous ADAM substrates.

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Preferential cleavage of CD44 homodimers.(A) CD44 dimers could be detected on denaturing SDS-PAGE gels under non-reducing conditions25. The uncleavable CD44 KR-MT mutant dimerizes only minimally. TPA did not influence dimerization but enhanced TPA-induced release of soluble CD44 ectodomain. CD44 wt or its KR-MT mutant were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. (A) samples were run on one gel, but the empty lane between −TPA and +TPA was removed. (B) The column diagram shows mean values of relative levels of ectodomain cleavage ± SD obtained by resolution on reducing gels from three independent experiments.
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f7: Preferential cleavage of CD44 homodimers.(A) CD44 dimers could be detected on denaturing SDS-PAGE gels under non-reducing conditions25. The uncleavable CD44 KR-MT mutant dimerizes only minimally. TPA did not influence dimerization but enhanced TPA-induced release of soluble CD44 ectodomain. CD44 wt or its KR-MT mutant were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. (A) samples were run on one gel, but the empty lane between −TPA and +TPA was removed. (B) The column diagram shows mean values of relative levels of ectodomain cleavage ± SD obtained by resolution on reducing gels from three independent experiments.

Mentions: Transmission of a conformational change through the plasma membrane cannot be achieved by single-pass transmembrane proteins. The intracellular protein kinase of RTKs for example is activated by ligand-induced relative positioning of individual subunits in receptor dimers in response to ligand binding (“outside-in” signaling) (reviewed in ref. 32).We hypothesized that “inside-out” signaling which confers substrate protease accessibility would also require dimerization. Both NRG1 and CD44 form homodimers which may represent an essential pre-condition for their regulated ectodomain cleavage25. Because CD44 dimers are stabilized by S-S bridges, part of CD44 migrated as dimer in a non-reducing gel. However, the un-cleavable CD44 mutant (KR-MT) did almost not dimerize at all (Fig. 7, lanes 2 and 3). TPA induced ectodomain cleavage products above a low basal level are detected in CD44 wt but only barely in case of the mutant KR-MT (Fig. 7, compare lanes 2 and 3 with lanes 5 and 6), correlating with trypsin accessibility reduction of CD44 KR-MT (Fig. 4A).


Growth factor and co-receptor release by structural regulation of substrate metalloprotease accessibility
Preferential cleavage of CD44 homodimers.(A) CD44 dimers could be detected on denaturing SDS-PAGE gels under non-reducing conditions25. The uncleavable CD44 KR-MT mutant dimerizes only minimally. TPA did not influence dimerization but enhanced TPA-induced release of soluble CD44 ectodomain. CD44 wt or its KR-MT mutant were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. (A) samples were run on one gel, but the empty lane between −TPA and +TPA was removed. (B) The column diagram shows mean values of relative levels of ectodomain cleavage ± SD obtained by resolution on reducing gels from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120278&req=5

f7: Preferential cleavage of CD44 homodimers.(A) CD44 dimers could be detected on denaturing SDS-PAGE gels under non-reducing conditions25. The uncleavable CD44 KR-MT mutant dimerizes only minimally. TPA did not influence dimerization but enhanced TPA-induced release of soluble CD44 ectodomain. CD44 wt or its KR-MT mutant were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. (A) samples were run on one gel, but the empty lane between −TPA and +TPA was removed. (B) The column diagram shows mean values of relative levels of ectodomain cleavage ± SD obtained by resolution on reducing gels from three independent experiments.
Mentions: Transmission of a conformational change through the plasma membrane cannot be achieved by single-pass transmembrane proteins. The intracellular protein kinase of RTKs for example is activated by ligand-induced relative positioning of individual subunits in receptor dimers in response to ligand binding (“outside-in” signaling) (reviewed in ref. 32).We hypothesized that “inside-out” signaling which confers substrate protease accessibility would also require dimerization. Both NRG1 and CD44 form homodimers which may represent an essential pre-condition for their regulated ectodomain cleavage25. Because CD44 dimers are stabilized by S-S bridges, part of CD44 migrated as dimer in a non-reducing gel. However, the un-cleavable CD44 mutant (KR-MT) did almost not dimerize at all (Fig. 7, lanes 2 and 3). TPA induced ectodomain cleavage products above a low basal level are detected in CD44 wt but only barely in case of the mutant KR-MT (Fig. 7, compare lanes 2 and 3 with lanes 5 and 6), correlating with trypsin accessibility reduction of CD44 KR-MT (Fig. 4A).

View Article: PubMed Central - PubMed

ABSTRACT

Release of cytokines, growth factors and other life-essential molecules from precursors by a-disintegrin-and-metalloproteases (ADAMs) is regulated with high substrate-specificity. We hypothesized that this is achieved by cleavage-regulatory intracellular-domain (ICD)-modifications of the precursors. We show here that cleavage-stimuli-induced specific ICD-modifications cause structural substrate changes that enhance ectodomain sensitivity of neuregulin-1 (NRG1; epidermal-growth-factor) or CD44 (receptor-tyrosine-kinase (RTK) co-receptor) to chymotrypsin/trypsin or soluble ADAM. This inside-out signal transfer required substrate homodimerization and was prevented by cleavage-inhibitory ICD-mutations. In chimeras, regulation could be conferred to a foreign ectodomain, suggesting a common higher-order structure. We predict that substrate-specific protease-accessibility-regulation controls release of numerous ADAM substrates.

No MeSH data available.


Related in: MedlinePlus