Biochemical Genetics of Signal Transduction

Mihaela Serpe, PhD, Head, Unit on Cellular Communication
Peter Nguyen, Biological Laboratory Technician

We investigate molecular mechanisms that regulate cell-cell communication during development and homeostasis. Cells communicate with each other by relying on a handful of conserved families of signaling molecules. The TGF-β superfamily of growth and differentiation factors, including TGF-βs/Activins and the bone morphogenetic proteins (BMP), is one of the largest of these families. TGF-β signaling factors have the ability to function as morphogens, that is, to specify cell fate in a concentration-dependent manner. In addition, TGF-β–type factors provide a mechanism for coupling a cell to its environment, endowing the cell with the necessary plasticity to respond appropriately to changes in its environment or even its own state. We use the Drosophila model system and a variety of molecular and biochemical approaches as a paradigm for cell-cell signaling mechanisms to study genes that modulate the function of TGF-β–type signals. We are interested in how the extracellular availability of TGF-β factors is regulated in time and space as well as in the role of the cell surface in modulating local activation of TGF-β–type signaling. We are working toward a genetic and biochemical description of how signaling is modulated and how it has been adapted for different roles during development and evolution.

The role of enzyme-substrate interactions in formation of BMP morphogen gradients

Nguyen, Serpe; in collaboration with Umulis, O’Connor

In the early Drosophila embryo, decapentaplegic (dpp), the gene encoding a BMP-type ligand, is transcribed uniformly throughout the dorsal domain, yet only about 10 percent of cells along the dorsal midline receive high levels of signal. In the pupal wing, Dpp diffuses from the longitudinal veins into the posterior cross-vein–competent zone and creates a corridor of peak signaling that is perpendicular to the source of morphogen. In both instances, the formation of the Dpp gradient occurs at a post-transcriptional level and involves modulation by additional secreted gene products.

In the early embryo, Dpp is bound in a complex containing Short gastrulation (Sog). The complex inhibits binding of Dpp to its receptors in lateral regions and facilitates long-range ligand diffusion, shuttling Dpp from the lateral domain toward the midline. An important component that helps create flux is the processing of Sog by Tolloid (Tld), a metalloprotease of the BMP-1 family. The net movement of Dpp dorsally is generated by reiterated cycles of complex formation, diffusion, and destruction of Sog by Tld. Drosophila has two highly homologous proteases of the BMP-1/Tld family, Tld and Tolloid-related (Tlr), which are both expressed in similar patterns in the early embryo and process Sog.

Drosophila has two highly homologous proteases of the BMP-1/Tld family, Tld and Tolloid-related (Tlr), which are both expressed in similar patterns in the early embryo In contrast, in the wing Sog needs to be processed by Tlr to enable proper Dpp signaling for posterior cross-vein development—Tld cannot take its place. We previously demonstrated that enzyme kinetics regulate developmental specificity, at least in part. Tlr processes Sog at least an order-of-magnitude more slowly than does Tld. It appears that, for both developmental processes, a balance between Sog’s inhibitory and positive activities is crucial for proper patterning. The Tlr catalytic properties fulfill the temporal constraints of pupal wing development while the more rapid kinetics of Tld are matched to the rapid development of the embryo.

In both the embryo and posterior cross-vein, Sog plays both positive and negative roles in regulating BMP activity, a phenomenon previously referred to as the Sog paradox. The negative role derives from Sog’s ability to block ligands’ access to receptors. The positive effect derives from Sog’s ability to facilitate Dpp diffusion. Interestingly, Chordin, the vertebrate orthologue of Sog, acts as an inhibitor only when expressed in Drosophila and cannot promote long-range Dpp signaling. At the molecular level, Sog and Chordin differ in that processing of Sog by Tld requires the BMP ligand as an obligatory co-substrate while Chordin does not. To determine the source of this difference, we modeled the Tld catalytic domain by using the crystal structure of crayfish Astacin, the founder member of this metalloprotease family. We purified and sequenced the Sog cleavage fragments, derived a consensus cleavage recognition sequence, and used the peptide to study the enzyme-substrate interactions in Sog for comparison with Chordin sequences. From this modeling, we hypothesized that several residues at the processing site might be responsible for making one, but not the other substrate dependent on BMP binding for processing. We successfully generated Sog variants that are BMP-independent Tld substrates in vitro. We are now characterizing their in vivo effect on BMP signaling.

Our working hypothesis is that Sog’s ability to function in a transport process as a long-range BMP agonist resides, in molecular terms, in the BMP’s co-substrate requirements for Tld-mediated Sog degradation. Computational modeling by our collaborator David Umulis supported this hypothesis, indicating that the co-substrate requirement for Sog processing by Tld is critical for proper Dpp gradient formation. Computations that relax this constraint and allow for Sog degradation when not complexed with Dpp show a great reduction in Dpp flux toward the dorsal midline.

Shaping BMP gradients through ligand-dependent modulation and positive feedback

Serpe; in collaboration with Blair, Umulis, O’Connor

BMP gradients in the early embryo and the pupal wing share similar features. They both form sharp boundaries and require two BMP-type ligands, one of which is Dpp. They differ in their spatial constraints and in their requirement for another BMP binding molecule, Crossveinless-2 (Cv-2). Cv-2 is crucial for BMP signaling in the posterior cross-vein; however, the null cv-2 alleles we created by targeted mutagenesis exhibit normal early embryonic patterning. Loss of Cv-2 causes loss of BMP signaling in the developing cross-veins while high levels of Cv-2 sequester ligands. As with Sog, Cv-2 exhibits both BMP agonistic and antagonist activities. In contrast to sog mutants, null cv-2 early embryos retain peak BMP signaling dorsally; in fact, at low temperatures, a slight enhancement of the signaling level occurs. Thus, Cv-2 appears to have an antagonistic effect on Dpp. Further analysis confirmed that Cv-2 has Dpp-antagonistic activity for the large range of Dpp and Cv-2 concentrations tested. Yet, in the pupal wing, BMP signaling shows a biphasic response to changes in Cv-2 levels: low signaling without Cv-2, maximum signaling with moderate levels of Cv-2, and then decreased signaling with elevated levels of Cv-2. Our studies indicate that Cv-2 behaviors are in fact ligand-dependent activities.

The cross-vein specification requires two BMP ligands, Dpp and Glass Bottom Boat (Gbb). Peak BMP signaling in the future cross-vein region could be attributable to Dpp and Gbb homo- and/or heterodimers. Cv-2 binds to BMP ligands but, unlike Sog, does not mediate the ligands’ long-range transport. We have shown that Cv-2 binds to the cell surface and heparan sulfate proteoglycans. In collaboration with Seth Blair, we demonstrated that Cv-2 acts locally within the cross-vein itself. It forms a transient complex with the BMP signaling molecule and the type I BMP receptor. Through this complex, Cv-2 can antagonize BMP ligands with high affinity for the receptor (such as Dpp) while facilitating the recruitment of low-affinity ligands (i.e., Gbb) in close proximity to the receptors, thereby potentiating their signaling. Consequently, Cv-2 can distinguish between various BMP ligands and differentially modulate their signaling. Our model of Cv-2 function shows how a cell-surface ligand-binding molecule can act locally to promote or inhibit signaling.

In addition, Cv-2 is a transcriptional target of BMP signaling. This positive feedback appears to play a role in refining the initially broad region of BMP signaling observed at early stages of posterior cross-vein development to the more tightly focused signaling observed at later stages. The formation of sharp boundaries is termed spatial bistability. In the case of Cv-2, mathematical modeling demonstrated that coupling the extracellular effects with positive feedback on the production of Cv-2 itself can lead to bistable signaling wherein a very sharp transition can be generated between cells that receive high versus low levels of signal. Therefore, the positive feedback of a cell-surface BMP-binding protein provides a mechanism for spatial refinement of signaling.

Together, our data indicate that, depending on the particular context in which it acts, the combined properties of Cv-2 enable the protein to have remarkably versatile effects on signaling and thus to tune BMP signaling exquisitely.

Serpe M, Umulis D, Ralston A, Chen J, Olson DJ, Avanesov A, Othmer H, O’Connor MB, Blair SS. The BMP-binding protein Crossveinless-2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila. Dev Cell 2008;14:940-953.

Collaborators

Seth Blair, PhD, University of Wisconsin, Madison, WI
Michael O’Connor, PhD, University of Minnesota and Howard Hughes Medical Institute, Minneapolis, MN
David Umulis, PhD, Purdue University, W. Lafayette, IN

For further information, contact serpemih@mail.nih.gov or visit http://ucc.nichd.nih.gov