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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.

No MeSH data available.


Related in: MedlinePlus

(A–C) ICDs modulate cleavage and protease accessibility of “foreign“ ectodomains in chimeras of CD44 and NRG1. (A) NRG1E/CD44(TM + ICD): CD44 ICD regulates cleavage of NRG1 ectodomain; effect of CD44 ICD mutants. See sketch for construction of chimeras. (B) Induced cleavage of NRG1E/CD44(TM + ICD) is inhibited by batimastat. (C) NRG1 wt and NRG1E/CD44(TM + ICD) show similar protease accessibility. (A,B) NRG wt and the NRG1E/CD44(TM + ICD) chimera were expressed in HEK293T cells E = ectodomain, TM = transmembrane domain, ICD = intracellular domain. The chimeric construct and its CD44 ICD mutants were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. V = vector control. Wt = wild type. Batimastat (10 μM) was added 15 min prior to TPA. In (A) upper panel the solNRG1E samples were run on one gel, however an empty lane without sample that separated −TPA and +TPA was removed. The samples of the middle and lower panels were run on the same gel. The samples of (B) were also run on one and the same gel but several lanes that showed other samples were removed. (C) Protease accessibility regulation by TPA in the presence of batimastat (10 μM) was determined as in Fig. 1B. Column diagrams show mean values of relative level of ectodomain cleavage ± SD from three independent experiments.
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f6: (A–C) ICDs modulate cleavage and protease accessibility of “foreign“ ectodomains in chimeras of CD44 and NRG1. (A) NRG1E/CD44(TM + ICD): CD44 ICD regulates cleavage of NRG1 ectodomain; effect of CD44 ICD mutants. See sketch for construction of chimeras. (B) Induced cleavage of NRG1E/CD44(TM + ICD) is inhibited by batimastat. (C) NRG1 wt and NRG1E/CD44(TM + ICD) show similar protease accessibility. (A,B) NRG wt and the NRG1E/CD44(TM + ICD) chimera were expressed in HEK293T cells E = ectodomain, TM = transmembrane domain, ICD = intracellular domain. The chimeric construct and its CD44 ICD mutants were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. V = vector control. Wt = wild type. Batimastat (10 μM) was added 15 min prior to TPA. In (A) upper panel the solNRG1E samples were run on one gel, however an empty lane without sample that separated −TPA and +TPA was removed. The samples of the middle and lower panels were run on the same gel. The samples of (B) were also run on one and the same gel but several lanes that showed other samples were removed. (C) Protease accessibility regulation by TPA in the presence of batimastat (10 μM) was determined as in Fig. 1B. Column diagrams show mean values of relative level of ectodomain cleavage ± SD from three independent experiments.

Mentions: To reveal such inducible structural changes, we probed the structural state of substrate ectodomains in presence or absence of cleavage stimuli, using accessibility to trypsin/chymotrypsin or soluble ADAM catalytic domain as a read-out. As substrates we used doubly-tagged molecules transfected into RPM-MC human pancreatic carcinoma cells (CD44) or HEK cells (NRG1). RPM-MC cells do not express CD44, permitting to examine overexpressed CD44 and its mutants without interference by wt endogenous counterparts. Both NRG1 and CD44 carried N-terminal FLAG tags; NRG1 and CD44 carried C-terminal c-myc tags or alternatively EGFP tags in select cases of NRG1 experiments (see schematics in Figs 2A, 3A and 6A). Surface expression of constructs was confirmed after transfection by FACS detection of the N′terminal FLAG ectodomain tag and by western blot detection of the N′terminal FLAG or C-terminal MYC tag (Fig. 1). For NRG1 there was no difference in surface expression whether a C-terminal c-myc or EGFP tag was used (data not shown). The surface expression shown here (Fig. 1) confirms results previously obtained by FACS analysis of transfected NRG1 expression contructs16. We tested regulated cleavage by endogenous ADAM for transfected CD44 or NRG1 and also for endogenous CD44 (Supplemental Fig. 1; MDA-MB-231 breast adenocarcinoma cells) and for endogenous NRG1 as indicated in the text below. Cleavage was induced by TPA or Angiotensin II (AngII, in HEK293T cells expressing the angiotensin-II-type1-receptor). For CD44 we chose trypsin because it produces only one cut close to the site of ADAM10 cleavage (schematic in Fig. 2A); other putative trypsin sites are apparently hidden inside CD44’s three-dimensional structure (schematic in Fig. 2B). For NRG1 we chose chymotrypsin. Chymotrypsin cuts NRG1 only once between F229 and Y230 (schematic in Fig. 3A)26. All putative ADAM17 cleavage sites reported are within the sequence 226MASFYKHLGIEFME239 surrounding the chymotrypsin site. The major cut in vivo is identical with that by chymotrypsin27. In these experiments, endogenous ADAM activity was blocked by batimastat (or GM6001), and γ-secretase was inhibited by DAPT, to exclude any other proteolysis (by γ-secretase and subsequent ICD processing) beyond the action of trypsin, chymotrypsin, or soluble ADAM.


Growth factor and co-receptor release by structural regulation of substrate metalloprotease accessibility
(A–C) ICDs modulate cleavage and protease accessibility of “foreign“ ectodomains in chimeras of CD44 and NRG1. (A) NRG1E/CD44(TM + ICD): CD44 ICD regulates cleavage of NRG1 ectodomain; effect of CD44 ICD mutants. See sketch for construction of chimeras. (B) Induced cleavage of NRG1E/CD44(TM + ICD) is inhibited by batimastat. (C) NRG1 wt and NRG1E/CD44(TM + ICD) show similar protease accessibility. (A,B) NRG wt and the NRG1E/CD44(TM + ICD) chimera were expressed in HEK293T cells E = ectodomain, TM = transmembrane domain, ICD = intracellular domain. The chimeric construct and its CD44 ICD mutants were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. V = vector control. Wt = wild type. Batimastat (10 μM) was added 15 min prior to TPA. In (A) upper panel the solNRG1E samples were run on one gel, however an empty lane without sample that separated −TPA and +TPA was removed. The samples of the middle and lower panels were run on the same gel. The samples of (B) were also run on one and the same gel but several lanes that showed other samples were removed. (C) Protease accessibility regulation by TPA in the presence of batimastat (10 μM) was determined as in Fig. 1B. Column diagrams show mean values of relative level of ectodomain cleavage ± SD from three independent experiments.
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f6: (A–C) ICDs modulate cleavage and protease accessibility of “foreign“ ectodomains in chimeras of CD44 and NRG1. (A) NRG1E/CD44(TM + ICD): CD44 ICD regulates cleavage of NRG1 ectodomain; effect of CD44 ICD mutants. See sketch for construction of chimeras. (B) Induced cleavage of NRG1E/CD44(TM + ICD) is inhibited by batimastat. (C) NRG1 wt and NRG1E/CD44(TM + ICD) show similar protease accessibility. (A,B) NRG wt and the NRG1E/CD44(TM + ICD) chimera were expressed in HEK293T cells E = ectodomain, TM = transmembrane domain, ICD = intracellular domain. The chimeric construct and its CD44 ICD mutants were transfected into RPM-MC cells and cleavage by TPA was analyzed as in Fig. 2. V = vector control. Wt = wild type. Batimastat (10 μM) was added 15 min prior to TPA. In (A) upper panel the solNRG1E samples were run on one gel, however an empty lane without sample that separated −TPA and +TPA was removed. The samples of the middle and lower panels were run on the same gel. The samples of (B) were also run on one and the same gel but several lanes that showed other samples were removed. (C) Protease accessibility regulation by TPA in the presence of batimastat (10 μM) was determined as in Fig. 1B. Column diagrams show mean values of relative level of ectodomain cleavage ± SD from three independent experiments.
Mentions: To reveal such inducible structural changes, we probed the structural state of substrate ectodomains in presence or absence of cleavage stimuli, using accessibility to trypsin/chymotrypsin or soluble ADAM catalytic domain as a read-out. As substrates we used doubly-tagged molecules transfected into RPM-MC human pancreatic carcinoma cells (CD44) or HEK cells (NRG1). RPM-MC cells do not express CD44, permitting to examine overexpressed CD44 and its mutants without interference by wt endogenous counterparts. Both NRG1 and CD44 carried N-terminal FLAG tags; NRG1 and CD44 carried C-terminal c-myc tags or alternatively EGFP tags in select cases of NRG1 experiments (see schematics in Figs 2A, 3A and 6A). Surface expression of constructs was confirmed after transfection by FACS detection of the N′terminal FLAG ectodomain tag and by western blot detection of the N′terminal FLAG or C-terminal MYC tag (Fig. 1). For NRG1 there was no difference in surface expression whether a C-terminal c-myc or EGFP tag was used (data not shown). The surface expression shown here (Fig. 1) confirms results previously obtained by FACS analysis of transfected NRG1 expression contructs16. We tested regulated cleavage by endogenous ADAM for transfected CD44 or NRG1 and also for endogenous CD44 (Supplemental Fig. 1; MDA-MB-231 breast adenocarcinoma cells) and for endogenous NRG1 as indicated in the text below. Cleavage was induced by TPA or Angiotensin II (AngII, in HEK293T cells expressing the angiotensin-II-type1-receptor). For CD44 we chose trypsin because it produces only one cut close to the site of ADAM10 cleavage (schematic in Fig. 2A); other putative trypsin sites are apparently hidden inside CD44’s three-dimensional structure (schematic in Fig. 2B). For NRG1 we chose chymotrypsin. Chymotrypsin cuts NRG1 only once between F229 and Y230 (schematic in Fig. 3A)26. All putative ADAM17 cleavage sites reported are within the sequence 226MASFYKHLGIEFME239 surrounding the chymotrypsin site. The major cut in vivo is identical with that by chymotrypsin27. In these experiments, endogenous ADAM activity was blocked by batimastat (or GM6001), and γ-secretase was inhibited by DAPT, to exclude any other proteolysis (by γ-secretase and subsequent ICD processing) beyond the action of trypsin, chymotrypsin, or soluble ADAM.

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