The first objective was to carry out a detailed structure-function analysis of activin A, a representative ligand of the TGF-beta superfamily, in order to identify amino acid residues or subdomains of the ligand which are important for biological activity and receptor binding. The second aim was to study several aspects, including structure-function, of type II serine/threonine kinase (STK) receptors for activins (ActRII). During the course of our work, the first sequences of type I receptors became available. This prompted us to reset objectives in the project and incorporate biochemical, descriptive and functional studies on type I receptors. Type I/type II heteromeric receptor complexes have been shown to bind ligand with high affinity, and type I receptors are essential in such receptor complexes for biological response to the ligands. We have verified the previously proposed specificity for ligand binding and signaling of the individual receptors in typeI/type II receptor complexes. The expression of the genes encoding activin A and B, activin type II receptors (ActRII-A and -B) and several type I receptors (ALK-2 and ALK-4 (also named ActRI-A and -B, respectively), and ALK-3 and ALK-6 (receptors for BMPs) has been studied during mouse embryogenesis, mainly by in situ hybridization. Using mutants of the type II and type I STK receptors, we then aimed at the manipulation of the activin signaling process in cell lines (such as ES and EC cells) and in the amphibian and mouse embryo. Finally, the cDNA cloning of vertebrate homologs (now called SMADs) of a signal transducer identified in Drosophila and named mothers against dpp (MAD), allowed us to initiate studies at the level of signal transduction as well. These started with the functional characterization of SMAD and its subdomains using overexpression in the amphibian embryo. It is our opinion that we have fulfilled the objectives which were put forward in the project proposal, and have anticipated the rapid evolution in this competitive field, as illustrated by our work on type I STK receptors and studies in signal transduction.
The main achievements in the project can be summarized as follows:
(i) the completion of all protocols and tools needed for this project. This includes the cloning and manipulation (tagging with a heterologous, non-interfering epitope, mutagenesis) of the murine homologs of type II and type I activin receptors; the construction of constitutively active type I receptors; the generation and characterization of anti-receptor antibodies; the improved large-scale purification of recombinant activin A produced in insect cells; the commercialization as research product of recombinant activin A by the SME partner; transient expression of combinations of transfected receptors;affinity-crosslinking with radio-iodinated ligand in cells; Scatchard analysis; the construction and expression of more than 40 mutants of activin A, and the analysis of their bioactivity in a panel of bioassays;
(ii) other achievements include: insight into the ligand-binding properties of and receptor complex formation between different full-length or truncated type I/type II receptors (one of the observations is that type IIA and IIB receptors tend to form homodimers rather than heterodimers); different forms of follistatin neutralize activin bio-activity by interference with its binding to type II receptors; the completion of the expression analysis of ligand subunits, type I and type II receptors, follistatin, and two other type I receptors for BMPs (ALK-3 and ALK-6); documentation of receptor isoform expression in cell lines; the finding that BMP-7 (OP-1) can also bind to ActR-II and signal through a variety of type I/type II receptor complexes containing ActR-II, including those containing ALK-3 and ALK-6;
(iii) ES and P19 EC cell lines stably transformed with (wild type and mutated variants of) ActR-IIB2, an interesting splice variant of ActR-IIB that is predominantly, if not exclusively, used by inhibin for competing with activins in several bioassays in which inhibin itself has no detectable direct effects;
(iv) cell lines, including ES cells, stably transformed with constitutively active variants of ActR-I(ALK-2 and ALK-4), silent truncated ALK-4 (ActRI-B) and ALK-6 (BMPRI-B) lacking the kinase domain, respectively, for use in transgenesis in the mouse; activation of the receptor synthesis in these cells resulting in overexpression of the truncated, non-signaling receptor by Cre recombinase has been obtained. The respective ES cells are now being used to make transgenic mice; the trALK-4 and trALK-6 mice will be intercrossed with selected strains of mice transgenic for Cre recombinase;
(v) selection and production of five interesting activin A mutant proteins, one of which is an agonist;
(vii) isolation of the cDNA encoding vertebrate homologs of Drosophila MAD, its identification (concurrently with others in the field) as a signal transducer, the functional characterization of subdomains of this polypeptide, the intramolecular regulation of the transactivation activity of the C-terminal domain by a segment of the (inhibitory) N-terminal domain, the development of functional assays that could be used for future screening of drugs that act at the level of SMAD-like polypeptides, the study of their expression pattern in vivo by in situ hybridization in the mouse and amphibian embryo;
(viii) the completion of a study initiated last year, suggesting that activin is an important regulator of neurogenesis in the mammalian embryo, as studied by differentiation of P19 EC cells in vitro.
MAJOR SCIENTIFIC BREAKTHROUGHS
Major breakthroughs were obtained in our work on activin type I and type II receptors (ligand binding and receptor oligomerization), the mechanism of neutralization of activin bioactivity by follistatins, the completion of the study of the expression patterns of all components of the activin ligand-receptor system during mouse embryogenesis, and the finding that ActRII is a candidate functional receptor for BMP-7 (OP-1) in diverse receptor complexes. We want to emphasize the breakthroughs in two other studies, one of which (activin mutants) could be achieved only by a strong and very efficient interaction between all partners, and our first studies on an important class of cytosolic signal tranducing polypeptides (SMAD proteins) which upon activation translocate to the nucleus, where they exert a function in regulation of gene expression. In particular, work in this project and by other groups on vertebrate members of the SMAD superfamily strongly suggests the tight link between regulatory mechnanisms in development (TGF-beta/receptor/SMAD pathways) and the tumor suppressor activity of SMAD genes. Part of the work described in the original project description will be finished soon or has enabled us to proceed into further important projects, some of which are briefly discussed below.
More than 40 mutants of activin have been constructed. A large panel (>30) are single residue substitutions (initially by alanine), a more limited panel are chimeric molecules between activin A and BMP-7/OP-1 and TGFbetas, respectively. The vaccinia virus-based expression system that we use yields sufficient quantities of these mutants to test them in in vitro assays (Xenopus animal cap assay, which is a rapid and sensitive assay, P19 EC cell aggregates, FSH assay in pituitary cells). We have been able to identify - for the first time in the TGFbeta ligand family - one single residue, the positive charge of which is essential for bioactivity. Moreover, we have identified in our collection one other mutant activin (in which only one amino acid was modified) that is an agonist. Five point mutants, selected according to the criteria listed above, have recently been produced in large amounts by our SME partner using a baculovirus-based transient expression system in insect cells, and are being purified according to the protocol established previously for wild type activin A. The ongoing work involves the use of these purified mutant activins and the comparison of their binding properties to type II and type I/type II receptors.
Truncated (tr) receptors interfere with signaling pathways in ES cells and P19 EC cells. For example, these were stably transformed with an expression vector encoding trActR-IIB2, from which the entire intracellular kinase domain was deleted. In the P19 EC cell clones, a clear correlation was found between the amount of expressed trActR-IIB2 and its (now inhibitory) effect on the induction of an activin-responsive 3TPLuc reporter construct. Another assay demonstrated that inhibition of retinoic acid-induced neuronal differentiation by activin was blocked in these trActR-IIB2 cell lines. Additional experiments strongly suggest that overexpressed trActR-IIB2 causes a dominant negative effect by outtitrating type I receptors, and thereby abolish activin binding to complexes containing the endogenous ActR-IIA. These EC cell clones are now important tools for studying aspects of in vitro differentiation which are relevant for murine embryogenesis.
ES cells have also been stably transformed with full-length ActR-IIB2, and will be tested for enhanced response to the ligand. Important aims of these studies are also to develop similar ES cell clones which can be used for transgenesis in the mouse. For studies with type I receptors, the following approaches are used: (i) in vitro made RNA from DNA encoding constitutively active variants (these have one aprticular amino acid change in the kinase domain) of the type I receptors ALK-2 and ALK-4 have been used in injections in the Xenopus embryo for comparison of the effect on the expression of known activin-modulated and/or mesodermal genes; (ii) the same receptors are stably transformed to EC and ES cells. Based on previously established in vitro characteristics, ES cellclones have been selected and are being introduced into host mouse embryos in order to study the role of the activin signaling pathway in early mouse development; (iii) a loss-of-function approach of ALK-4 (ActR-IB) and ALK-6, using Cre-mediated activation of overexpression of trALK-4 and trALK-6 in specific tissues or cell types at later stages of development in the mouse, in particular since our own data also suggest that these signaling pathways are important for organogenesis.
Funding SchemeCSC - Cost-sharing contracts
NW7 1AA London
3584 CT Utrecht