Herrlich Lab - Contributions to Science

Over more than 20 years we have had a sustained interest in signal transduction of G-protein coupled receptors and receptor tyrosine kinases, in particular the epidermal-growth-factor receptor (EGFR).

1. GPCR signal transduction: Dual coupling

During his thesis work Andreas Herrlich studied coupling of GPCRs to particular signal transduction pathways in epithelial cells. His results challenged the premise that GPCRs can only couple to one G-protein and therefore only activate one signaling pathway. Using direct G-protein labeling techniques, he showed that GPCRs can indeed couple to more than one G-protein (often two=dual coupling) and that each G-protein subunit (G-proteins contain α subunits and βγ-subunits) can signal independently. He also showed that dual coupling can depend on the level of GPCR ligand present, identifying an important switch on the Luteinizing hormone (LH) receptor that allows LH to stimulate differential cellular programs based on fluctuations of LH during the menstrual cycle which cause coupling to different G-proteins.

a.     Herrlich, A., Kühn, Grosse, R., Schmid, A, Schultz, G., and Gudermann, T. (1996) Involvement of Gs and Gi proteins in dual coupling of the luteinizing hormone receptor to adenylyl cyclase and phospholipase  C. The Journal of Biological Chemistry, 271,  16764-16772; PMID: 8663226 (139 citations).

b.     Grosse, R., Schmid, A, Schöneberg, T., Herrlich, A., Muhn, P., Schultz, G., and Gudermann, T. (2000) Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. The Journal of Biological Chemistry, 275, 9193-9200; PMID: 10734055 (130 citations).

2. Transactivation of EGFR

The mechanism of mitogen-activated-protein-kinases (MAPK) activation by GPCRs was an intensively researched area in the 1990s, after it was recognized that not only receptor tyrosine kinases (RTKs) like the EGFR can activate cellular growth pathways via MAPKs, but also GPCRs. The main finding of Andreas Herrlich's thesis work at the Free University Berlin was that GPCR-mediated MAPK activation requires an important intermediary step: Activation of the epidermal-growth-factor-receptor (EGFR). This phenomenon has been termed "EGFR transactivation" and is linked to GPCR-induced metalloprotease ADAM17 release of active EGF ligands from their transmembrane pro-forms. In this process the ectodomain of the pro-form is released and represents the active ligand (ectodomain shedding). The group of Axel Ullrich at the Max Planck lnstitute for Biochemistry, Munich discovered transactivation at the same time (Daub et al. Nature 1996). Andreas Herrlich's work resulted in two first author publications and in collaborative publications with the Ullrich Laboratory. Over the last 20 years literally hundreds if not thousands of publications in various fields of biology have confirmed findings on EGFR transactivation and it has now been recognized as one of the most important signaling hubs that links various cellular stimuli to cellular growth regulation as well as to the regulation of many other specialized functions of the cell. As example, during Andreas Herrlich's time as an Internal Medicine Resident and Chief Resident at Johns Hopkins and while working as a postdoctoral researcher in Peter Agre's and Landon King's Laboratory, he discovered that osmotic induction of the aquaporin water channel AQP5 in epithelial cells is mediated by EGFR transactivation involving the EGF ligand NRG1.

a.     Daub, H., Wallasch, C., Lankenau, A., Herrlich, A., and Ullrich, A. (1997) Signal characteristics of G protein-transactivated EGF receptor. EMBO Journal, 16, 7032-7044; PMID: 9384582 (690 citations).

b.     Herrlich, A., Daub, H., Knebel, A., Herrlich, P., Ullrich, A., Schultz, G., and Gudermann, T. (1998) Ligand­independent activation of platelet-derived growth factor receptor is a necessary intermediate in lysophosphatidic, acid-stimulated mitogenic activity in L cells. Proceedings of the National Academy of Sciences of the United States of America, 95, 8985-8990; PMID: 9671791 (165 citations)

c.      Grosse, R., Rölle, S., Herrlich, A., Hohn, J., and Gudermann, T. (2000) Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone. The Journal of Biological Chemistry, 275, 12251- 12260; PMID: 10766863 (73 citations).

d.    Herrlich, A., Leitch, V., and King, L. (2004) Role of proneuregulin 1 cleavage and human epidermal growth factor receptor activation in hypertonic aquaporin induction. Proceedings of the National Academy of Sciences of the United States of America, 101, 15799-15804; PMID: 15498868 (17 citations).

3. Regulation of ectodomain shedding

The Herrlich laboratory's primary expertise lies in the study of regulation of ectodomain cleavage and of its cell biological, physiological and pathophysiological consequences. Induced ectodomain cleavage by metalloproteases is carried out mainly by a-disintegrin-and-metalloprotease (ADAM) 10 or 17. Based on mouse experiments in ADAM17 hypomorph mice and on observations in humans with ADAM17 deletion, the physiologically most relevant ADAM17 substrates in vivo are EGF ligands and tumor-necrosis-factor-alpha (TNFα), a key inflammatory cytokine. However, ADAM17 and ADAM10 have many more in vitro identified substrates (>100), and it remains unclear how substrate specificity of cleavage is achieved. Such control is critically important, since altered cleavage of many ADAM10/17 substrates has been linked to human diseases, including inflammation, cancer, Alzheimer's, heart and kidney disease.

How ectodomain cleavage is regulated has been hotly debated for many years. It is regulated by intracellular signaling pathways, yet how these pathways connect to metalloprotease cleavage is unknown. It was widely assumed in the field that the metalloprotease represents the main and only target of intracellular signaling induced cleavage regulation, although the C-termini of ADAM10/17 can be removed without consequence for induced cleavage (our work and the work of others). However, we showed that substrates undergo specific and differential cleavage regulation via modification of their C-termini (Herrlich FASEB 2008, Dang JBC 2011), and identified signaling components involved in this regulation, using a large-scale shRNA screen directed against all human kinases and phosphatases (collaboration with Broad Institute, Cambridge, MA). In further work we used our screen data to identify specific cleavage regulatory signaling modules that regulate the cleavage of only particular ADAM substrates (Dang PNAS 2013). In a collaboration with Douglas Lauffenburger's Laboratory at MIT, we developed a novel assay that can measure metalloprotease activity in vitro or on living cells (PRAMA) and allows to distinguish between ADAM17, ADAM10 or several other MMP metalloprotease activities at the same time (Miller Integrative Biology 2011). Using this assay, we showed that metalloprotease activity was not significantly modified by cleavage stimuli, and that cleavage inhibited cells (by knockdown of our signaling modules) showed normal protease activity (Outstanding New Investigator Award Gordon Conference on Metalloproteases 2011; Dang PNAS 2013). Our PNAS paper was featured as "Editor's Choice" in Science Signaling. We thus challenged the status quo in the metalloprotease field by suggesting that cleavage regulation occurs to a large degree on the substrate level, next to some already known regulation on the metalloprotease level (Hartmann TIBS 2013; Parra MCB 2015).

a.     Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes. The FASEB Journal, 22, 4281-4295; PMID: 18757500.

b.     Dang, M., Dubbin, K., D'Aiello, A., Hartmann, M., Lodish, H., and Herrlich, A. (2011) Epidermalgrowth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs)  involves different protein kinase C (PKC) isoenzymes depending on the stimulus. The Journal of Biological Chemistry, 286, 17704-17713; PMID: 21454702.

c.     Miller, M. A., Barkal, L., Jeng, K., Herrlich, A., Moss, M., Griffith, L. G., and Lauffenburger, D. A. (2011) Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities. Integrative Biology, 3, 422-438; PMID: 21180711.

d.     Dang, M., Armbruster, N., Miller, M. A., Cermeno, E., Hartmann, M., Bell, G. W., Root, D. E., Lauffenburger, D. A, Lodish, H. F., and Herrlich, A. (2013) Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways. Proceedings of the National Academy of Sciences of the United States of America, 110, 9776-9781; PMID: 23720309. Editor's choice Science Signaling http://stke.sciencemag.org/content/6/280/ec140

a.     Wilson J.L., Kefaloyianni E., Stopfer L., Harrison C., Sabbisetti V., Fraenkel E., Lauffenburger D.A.*, Herrlich, A. (2017) Functional Genomics Approach Identifies Novel Signaling Regulators of TGFα Ectodomain Shedding. Molecular Cancer Research, PMID not available yet.

In the last few years we have worked intensively on understanding the molecular mechanisms of ectodomain shedding that allow substrate-specific signal transfer from the inside of the cell, via the C-terminus of the substrate, to the cell surface where metalloprotease cleavage occurs. We found that specific C-terminal modification of substrates induces a structural change of the substrate's ectodomain that allows metalloprotease access and cleavage ("Protease Accessibility Regulation"; Parra Scientific Reports 2016), and that this regulation requires substrate dimerization (Hartmann JBC 2015; "JBC Paper of the Year 2015" in Signal Transduction Category). C-terminal modification presumably modifies the relative positioning of the dimerization partners to each other and translates C-terminal modification into ectodomain protease accessibility. This completely novel cleavage regulatory mechanism would explain how different substrates cleaved by the same metalloprotease could be addressed in a substrate-specific manner. As examples, cancer cell proliferation/migration is dependent on C-terminal regulation of CD44 cleavage by the tumor suppressor merlin (Nf2) (Hartmann Mol Cancer Research 2015), featured in the AACR must read list 2015. Further, cleavage regulation of the EGF ligand NRG1 is mediated by C-terminal PKCδ-induced phosphorylation and affects axonal growth/myelination "in vivo" in trigeminal ganglion explants (Parra MCB 2015).

a.     Hartmann, M., Parra, L. M., Ruschel, A, Lindner, C., Morrison, H., Herrlich, A.*, and Herrlich, P*. (2015) Inside-out Regulation of Ectodomain Cleavage of Cluster-of-Differentiation-44 [CD44] and of Neuregulin-1 requires Substrate Dimerization. The Journal of Biological Chemistry, PMID: 25925953. *co-senior authors.            "JBC Paper of the week" and "JBC Paper of the Year 2015" in the Signal Transduction Category

b.     Hartmann, M., Parra, L. M., Ruschel, A, Behme, S., Li, Y., Morrison, H., Herrlich,  A.*,  and  Herrlich,  P.* (2015) Tumor Suppressor NF2BlocksCellularMigrationbyInhibitingEctodomainCleavage of  CD44. Molecular Cancer Research : MGR, PMID: 25652588. *co-senior authors                                                                        Featured on the AACR “Must Read List” 2015

c.     Parra L., Hartmann M., Schubach S., Yong L., Herrlich P.* and Herrlich A.* (2015) Distinct ICD substrate modifications selectively regulate ectodomain cleavage of NRG1 or CD44. Molecular and Cellular Biology; PMID: 26217011 *co-senior authors

d.     Parra L., Hartmann M., Schubach S., Yong L., Herrlich P.* and Herrlich A.* (2016) Inside-out intracellular signal induced regulation of ectodomain metalloprotease accessibility of NRG1 and CD44. Scientific Reports, accepted November 2016, no PMID available yet. *co-senior authors

4. Role of ADAM17 and its substrates in kidney fibrosis

Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of A-Disintegrin-And-Metalloprotease-17 (ADAM17), of its cleavage-activated pro-ligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG), as well as of their receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of pro-inflammatory and pro-fibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorph mice, specific ADAM17 inhibitor treated wt mice or mice with inducible knockout of ADAM17 in proximal tubule (Slc34a1-Cre) were very significantly protected against these effects. In vitro, in proximal tubule cells, we showed that AREG has unique pro-fibrotic actions that are potentiated by TNFα-induced AREG cleavage and depend on the transcriptional regulator YAP1. In vivo, in AKI and chronic-kidney-disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a novel therapeutic target for human kidney fibrosis. Using AREG injections into ADAM17 proximal tubule knockout mice, and mice with AREG knockout in the proximal tubule, we showed that proximal tubule-derived AREG is sufficient and necessary for the induction of kidney fibrosis after injury. With the help of the NIDDK Fibrosis Consortium (associate membership), we are now studying soluble AREG as a diagnostic or prognostic fibrosis biomarker in the serum and urine of two large clinical cohorts of CKD patients, the Boston Kidney Biopsy Study and the Seattle Kidney Study. In addition, we are studying AREG in 4000 patients of CRIC (Chronic Renal Insufficiency Cohort), in the context of an Ancillary Study to CRIC.  

a.   Kefaloyianni E, Muthu LM, Kaeppler J, Sun X, Sabbisetti V, Chalaris A, Rose-John S, Wong E, Sagi I, Waikar SS, Rennke H, Humphreys BD, Bonventre JV., Herrlich A.* (2016) ADAM17 substrate release in proximal tubule drives kidney fibrosis. Journal of Clinical Investigation Insight, 18;1(13). pii: e87023; PMID: 27642633.

b.   Kefaloyianni E.1, Raja Keerthi Raja M.1*, Schumacher J.1, Muthu ML.1†, Krishnadoss V.1‡, Waikar SS2, Herrlich A1 (2019) “Proximal tubule-derived amphiregulin amplifies and integrates profibrotic EGFR signals in kidney fibrosis” Journal of the American Society of Nephrology 2019, accepted August 15, 2019.

Reviews, Chapters, Monographs and Editorials:

  1. Herrlich A. Antineutrophil cytoplasmic antibodies (ANCA) in the long-term management of Wegener’s disease: How should we use them? Nephrology Rounds, 2009, Vol 11-6

  2. Herrlich A. Proteases. In: Lodish H, Berk A, Kaiser CA, Krieger, M, editors. Molecular Cell Biology, 7th edition (W. H. Freeman & Co., New York), 2011.

  3. Hartmann M., Herrlich A.*, Herrlich P.* Who decides when to cleave an ectodomain?. Trends in Biochemical Sciences January 2013 (* co-corresponding authors).

  4. Herrlich A. Proteases. In: Lodish H, Berk A, Kaiser CA, Krieger, M, editors. Molecular Cell Biology, 8th edition (W. H. Freeman & Co., New York), 2015.

Complete list of our published work in My Bibliography on Google Scholar

https://scholar.google.com/citations?hl=en&user=oPc89AkAAAAJ&view_op=list_works&sortby=pubdate ; http://www.ncbi.nlm.nih.gov/sites/myncbi/1JW2i5_-vRxkk/bibliography/40955963/public/?sort=date&direction=ascending