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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

Cellular Expression and surface localization of CD44 and NRG1 constructs.HEK cells were co-transfected with a plasmid encoding RFP and one of the constructs listed. All these constructs carried an N-terminal FLAG tag and a C-terminal MyC tag. The cultures were incubated in the presence of ADAM and Gamma secretase inhibition. After 24 hours one aliquot of each culture was analyzed by FACS using anti-FLAG antibodies and Alexa-488-labelled secondary antibodies. For CD44wt and CD44KR-MT we show two representative FACS plots (upper left) and for the remainder of the constructs we show surface fluorescent intensity values as determined by FACS (Table column 3). These plots and values indicate the presence of the ectodomain of all studied constructs on the cell surface. Another aliquot of each culture was taken for lysis and Western blotting using either antibodies against the N-terminal FLAG or C-terminal MYC tag. The blots were quantified by Image J, the intensities normalized for actin. Western blot band intensities normalized for actin are shown in Table column 4 and normalization of these values to respective wt controls is shown in Table column 5 and also in the column diagram (upper right). Both MYC and FLAG data from the Western blots indicate proper expression of all studied constructs.
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f1: Cellular Expression and surface localization of CD44 and NRG1 constructs.HEK cells were co-transfected with a plasmid encoding RFP and one of the constructs listed. All these constructs carried an N-terminal FLAG tag and a C-terminal MyC tag. The cultures were incubated in the presence of ADAM and Gamma secretase inhibition. After 24 hours one aliquot of each culture was analyzed by FACS using anti-FLAG antibodies and Alexa-488-labelled secondary antibodies. For CD44wt and CD44KR-MT we show two representative FACS plots (upper left) and for the remainder of the constructs we show surface fluorescent intensity values as determined by FACS (Table column 3). These plots and values indicate the presence of the ectodomain of all studied constructs on the cell surface. Another aliquot of each culture was taken for lysis and Western blotting using either antibodies against the N-terminal FLAG or C-terminal MYC tag. The blots were quantified by Image J, the intensities normalized for actin. Western blot band intensities normalized for actin are shown in Table column 4 and normalization of these values to respective wt controls is shown in Table column 5 and also in the column diagram (upper right). Both MYC and FLAG data from the Western blots indicate proper expression of all studied constructs.

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
Cellular Expression and surface localization of CD44 and NRG1 constructs.HEK cells were co-transfected with a plasmid encoding RFP and one of the constructs listed. All these constructs carried an N-terminal FLAG tag and a C-terminal MyC tag. The cultures were incubated in the presence of ADAM and Gamma secretase inhibition. After 24 hours one aliquot of each culture was analyzed by FACS using anti-FLAG antibodies and Alexa-488-labelled secondary antibodies. For CD44wt and CD44KR-MT we show two representative FACS plots (upper left) and for the remainder of the constructs we show surface fluorescent intensity values as determined by FACS (Table column 3). These plots and values indicate the presence of the ectodomain of all studied constructs on the cell surface. Another aliquot of each culture was taken for lysis and Western blotting using either antibodies against the N-terminal FLAG or C-terminal MYC tag. The blots were quantified by Image J, the intensities normalized for actin. Western blot band intensities normalized for actin are shown in Table column 4 and normalization of these values to respective wt controls is shown in Table column 5 and also in the column diagram (upper right). Both MYC and FLAG data from the Western blots indicate proper expression of all studied constructs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Cellular Expression and surface localization of CD44 and NRG1 constructs.HEK cells were co-transfected with a plasmid encoding RFP and one of the constructs listed. All these constructs carried an N-terminal FLAG tag and a C-terminal MyC tag. The cultures were incubated in the presence of ADAM and Gamma secretase inhibition. After 24 hours one aliquot of each culture was analyzed by FACS using anti-FLAG antibodies and Alexa-488-labelled secondary antibodies. For CD44wt and CD44KR-MT we show two representative FACS plots (upper left) and for the remainder of the constructs we show surface fluorescent intensity values as determined by FACS (Table column 3). These plots and values indicate the presence of the ectodomain of all studied constructs on the cell surface. Another aliquot of each culture was taken for lysis and Western blotting using either antibodies against the N-terminal FLAG or C-terminal MYC tag. The blots were quantified by Image J, the intensities normalized for actin. Western blot band intensities normalized for actin are shown in Table column 4 and normalization of these values to respective wt controls is shown in Table column 5 and also in the column diagram (upper right). Both MYC and FLAG data from the Western blots indicate proper expression of all studied constructs.
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