Dimerization and activation of the kit receptor by monovalent and bivalent binding of the stem cell factor.

The protooncogene c-kit encodes a tyrosine kinase receptor for the stem cell factor (SCF). Mutants of c-kit were shown to confer a pleiotropic defective phenotype and often display negative dominance in heterozygous mice. To explore the involvement of receptor dimerization in this genetic phenomenon, we employed both a human ligand, which does not recognize the murine receptor, and a rodent SCF, which binds to the human receptor with 100-fold reduced affinity as compared with human SCF. SCF binding to living cells was found to induce rapid and complete receptor dimerization that involved activation of the catalytic tyrosine kinase function. Although receptor dimerization can be attributed to the dimeric nature of the ligand, no dissociation of Kit dimers occurred at high excess of SCF, suggesting that receptor-receptor interactions are also involved in dimer stabilization. This was supported by in vitro formation of heterodimers between the human and murine Kit proteins through monovalent binding of species-specific human SCF. By coexpression of human and mouse Kit in murine fibroblasts, we found that receptor heterodimerization in living cells involved an increase in the affinity of human Kit for rat SCF and also an accelerated rate of receptor down-regulation. When a human Kit mutant lacking the kinase insert domain was coexpressed with the murine wild-type receptor, we observed a significant decrease in both the activation of the intact tyrosine kinase and its coupling to an effector protein, namely phosphatidylinositol 3'-kinase. Our results favor a receptor activation model that assumes an initial step of monovalent ligand binding, followed by an intermediate receptor dimer bound by one arm of the ligand molecule. This model predicts the existence of an intrinsic receptor dimerization site and provides a structural basis for genetic dominance of mutant SCF receptors.     

The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade.

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.     

Changes in Conformation and Stability upon SCF/sKit Complex Formation

Stem cell factor (SCF) is thought to be a member of the four-helical bundle cytokine superfamily, and exists in solution as a noncovalent homodimer. It is the ligand for Kit, a tyrosine kinase type III receptor. The interaction of SCF and Kit affects early hematopoietic progenitors, as well as gametocytes, melanocytes, and mast cells. Upon binding of SCF the Kit undergoes dimerization and transphosphorylation. Circular dichroism (CD), intrinsic fluorescence, and Fourier transform infrared (FTIR) spectroscopy were used for conformational analyses of free SCF, soluble Kit (sKit), and the complex. The sKit consisted of the extracellular domain of Kit, contained five Ig-like domains, and was prepared from the conditioned media of transfected Chinese hamster ovary cells. With these techniques, a reproducible conformational change was seen upon ligand/receptor binding. The far-UV CD and FTIR spectroscopy indicated a slight increase in the -helical content. The near-UV CD and fluorescence spectra showed changes in the environments of the aromatic amino acids. The thermal denaturation of SCF was not affected by complex formation, while the melting temperature of sKit increased only a few degrees when binding SCF. This indicates that binding is temperature dependent, consistent with titration calorimetry results published previously which demonstrated that there is a large enthalpy of binding. The conformational changes which accompany SCF/sKit binding could play a role in the receptor dimerization and signal transduction which follow.     

The I Domain is Essential for Echovirus 1 Interaction with VLA-2

VLA-2, the α2β1 integrin, mediates cell adhesion to collagen and laminin, and is the receptor for the human pathogen echovirus 1. Because of its similarity to domains present in other proteins that interact with collagen, a 191 amino acid region within the α2 subunit (the I domain) has been proposed as a potential site for ligand interactions. Although the α2 subunits of human and murine VLA-2 are 84% identical, human α2 promotes virus binding whereas murine α2 does not. We used murine/human chimeric α2 molecules to identify regions of the human molecule essential for virus binding. Virus bound efficiently to a chimeric protein in which the human I domain was inserted into murine α2, indicating that the human I domain is responsible for specific virus interactions. Monoclonal antibodies that inhibited virus attachment all recognized epitopes within the human I do-main, further suggesting that virus interacts with this portion of the molecule. Similarly, antibodies that prevented VLA-2-mediated cell adhesion to collagen also mapped to the I domain. These results indicate that the I domain plays a role in VLA-2 interactions both with virus and with extracellular matrix ligands.     

Chimeric interferon-gamma receptors demonstrate that an accessory factor required for activity interacts with the extracellular domain.

We determined the species specificity and function of structural domains of the interferon-gamma receptor (IFN-gamma R) by construction of human/murine chimeric IFN-gamma R cDNA clones and their expression in various cells. We demonstrate that we can reconstitute a biologically active IFN-gamma R in eukaryotic cells with chimeric receptors as long as the extracellular domain and an accessory factor are from the same species. These results indicate that the extracellular domain of the receptor interacts directly or indirectly with the species-specific accessory factor.     

A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion

The selectin family of adhesion molecules mediates the initial interactions of leukocytes with endothelium. The extracellular region of each selectin contains an amino-terminal C-type lectin domain, followed by an EGF-like domain and multiple short consensus repeat units (SCR). Previous studies have indirectly suggested a role for each of the extracellular domains of the selectins in cell adhesion. In this study, a panel of chimeric selectins created by exchange of domains between L- and P-selectin was used to directly examine the role of the extracellular domains in cell adhesion. Exchange of only the lectin domains between L- and P-selectin conferred the adhesive and ligand recognition functions of the lectin domain of the parent molecule. However, chimeric selectins which contained both the lectin domain of L- selectin and the EGF-like domain of P-selectin exhibited dual ligand- binding specificity. These chimeric proteins supported adhesion both to myeloid cells and to high endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo. Exchange of the SCR domains had no detectable effect on receptor function or specificity. Thus, the EGF- like domain of P-selectin may play a direct role in ligand recognition and leukocyte adhesion mediated by P-selectin, with the lectin plus EGF- like domains collectively forming a functional ligand recognition unit.     

A recombinant ectodomain of the receptor for the stem cell factor (SCF) retains ligand-induced receptor dimerization and antagonizes SCF-stimulated cellular responses.

The stem cell factor (SCF) is a polypeptide ligand that is essential for the development of germ cells, hematopoietic progenitor cells, and melanocyte precursors. It binds to a tyrosine kinase membrane receptor that is encoded by the c-kit proto-oncogene. We have constructed an expression vector that directs the synthesis of the entire extracellular ligand-binding domain of the Kit/SCF receptor. When expressed and amplified in Chinese hamster ovary cells, a secreted 90-kDa glycoprotein could be harvested from the growth medium of the cells in a soluble form. This extracellular portion of the Kit/SCF receptor, denoted Kit-X, was recognized by antibodies specific to the SCF receptor; and when injected into animals, it raised antibodies that were reactive with the complete membrane form of the receptor. Direct binding and covalent cross-linking of radiolabeled SCF showed that Kit-X fully retained high affinity ligand binding and also underwent efficient dimerization in the presence of the ligand. The capacity of Kit-X to act as an antagonist of SCF was assayed on cultured cells that overexpress the receptor. Simultaneous addition of SCF and Kit-X to these cells resulted in a stoichiometric inhibition of SCF binding and a consequent decrease in autophosphorylation of the SCF receptor on tyrosine residues. The inhibition extended to later SCF-mediated responses, including the association of the receptor with phosphatidylinositol 3'-kinase and coupling to the Raf1 protein kinase. These results indicate that the recombinant ectodomain of the Kit-SCF receptor can be used as a specific antagonist of SCF actions and may enable detailed molecular analysis of ligand-receptor interactions.     

Mapping ligand binding domains in chimeric fibroblast growth factor receptor molecules. Multiple regions determine ligand binding specificity

Fibroblast growth factors (FGFs) mediate essential cellular functions by activating one of four alternatively spliced FGF receptors (FGFRs). To determine the mechanism regulating ligand binding affinity and specificity, soluble FGFR1 and FGFR3 binding domains were compared for activity. FGFR1 bound well to FGF2 but poorly to FGF8 and FGF9. In contrast, FGFR3 bound well to FGF8 and FGF9 but poorly to FGF2. The differential ligand binding specificity of these two receptors was exploited to map specific ligand binding regions in mutant and chimeric receptor molecules. Deletion of immunoglobulin-like (Ig) domain I did not effect ligand binding, thus localizing the binding region(s) to the distal two Ig domains. Mapping studies identified two regions that contribute to FGF binding. Additionally, FGF2 binding showed positive cooperativity, suggesting the presence of two binding sites on a single FGFR or two interacting sites on an FGFR dimer. Analysis of FGF8 and FGF9 binding to chimeric receptors showed that a broad region spanning Ig domain II and sequences further N-terminal determines binding specificity for these ligands. These data demonstrate that multiple regions of the FGFR regulate ligand binding specificity and that these regions are distinct with respect to different members of the FGF family.     

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

The fifth immunoglobulin-like domain of the Kit receptor is required for proteolytic cleavage from the cell surface

Stem cell factor (SCF) initiates its biological effects by binding to its receptor Kit. Cell surface Kit is proteolytically cleaved to generate soluble Kit. Structure-function analysis of the extracellular region of Kit has implicated the first three immunoglobulin-like domains in SCF binding, and the fourth immunoglobulin-like domain in receptor dimerization. However, the role of the fifth immunoglobulin-like domain is unknown. To test the hypothesis that the fifth immunoglobulin-like domain is important for proteolytic cleavage of Kit from the cell surface, we constructed a mutant form of Kit in which the first four immunoglobulin-like domains are linked to the transmembrane and cytoplasmic domains (designated Kit-Del5). Kit-wild type (Kit-WT) and Kit-Del5 were expressed in the murine mast cell line IC2. Flow cytometry demonstrated that both Kit-WT and Kit-Del5 are displayed on the IC2 cell surface, and immunoblotting confirmed the presence of Kit proteins of the expected molecular weights, 154 kDa and 134 kDa, respectively. Although IC2-Kit-WT cells proteolytically cleave cell surface Kit, generating a 98 kDa soluble form of Kit, IC2-Kit-Del5 cells do not. These findings demonstrate that the fifth immunoglobulin-like domain of Kit is required for proteolytic cleavage of Kit from the cell surface.     

The first Ig domain of KIR3DL1 contacts MHC class I at a secondary site

KIR3DL1 is a highly polymorphic inhibitory killer cell Ig-like receptor (KIR) implicated in resistance to viral diseases such as AIDS. KIR3DL1 contains three Ig domains and is specific for MHC class I (MHC-I) molecules belonging to the HLA-Bw4 serogroup. The receptor's second and third Ig domains confer the Bw4 specificity, but the role of the first Ig domain (D0) in ligand recognition has remained enigmatic. We found that KIR3DL1 expressed in YTS cells and as a soluble receptor can weakly recognize additional MHC-I molecules including HLA-B*0702 and HLA-G. This interaction is highly sensitive to blocking with Abs to the MHC-I α3-domain and the anti-KIR3DL1 Ab Z27, but not the canonical blocking Ab DX9. Using chimeric receptors between KIR3DL1 and KIR2DL1 expressed on YTS cells and as soluble Fc-fusion proteins, we show that the D0 domain confers the broad functional recognition and binding as well as the reactivity with Z27. These results suggest that the presence of a second and independent site of interaction between D0 and MHC-I and that MHC-I could bridge KIR3DL1 molecules together in a manner that facilitates signaling.     

Structural implications of Siglec-5-mediated sialoglycan recognition

Sialic acid (Sia) Ig-like binding lectins are important mediators of recognition and signaling events among myeloid cells. To investigate the molecular mechanism underlying sialic acid Ig-like lectin (Siglec) functions, we determined the crystal structure of the two N-terminal extracellular domains of human myeloid cell inhibitory receptor Siglec-5 (CD170) and its complexes with two sialylated carbohydrates. The native structure revealed an unusual conformation of the CC′ ligand specificity loop and a unique interdomain disulfide bond. The α(2,3)- and α(2,6)-sialyllactose complexed structures showed a conserved Sia recognition motif that involves both Arg124 and a portion of the G-strand in the V-set domain forming β-sheet-like hydrogen bonds with the glycerol side chain of the Sia. Only few protein contacts to the subterminal sugars are observed and mediated by the highly variable GG′ linker and CC′ loop. These structural observations, in conjunction with surface plasmon resonance binding assays, provide mechanistic insights into linkage-dependent Siglec carbohydrate recognition and suggest that Siglec-5 and other CD33-related Siglec receptors are more promiscuous in sialoglycan recognition than previously understood.     

Molecular characteristics of IgA and IgM Fc binding to the Fcalpha/muR

Fcalpha/mu receptor (Fcalpha/muR), a novel Fc receptor for IgA and IgM, is a type I transmembrane protein with an immunoglobulin (Ig)-like domain in the extracellular portion. Although IgA and IgM bind to Fcalpha/muR, the molecular and structural characteristics of the ligand-receptor interactions have been undetermined. Here, we developed twelve monoclonal antibodies (mAbs) against murine Fcalpha/muR by immunizing mice deficient in Fcalpha/muR gene. Eight mAbs totally or partially blocked IgA and IgM bindings to Fcalpha/muR. These blocking mAbs bound to a peptide derived from the Ig-like domain of murine Fcalpha/muR, which is conserved not only in human and rat Fcalpha/muR but also in polymeric Ig receptor (poly-IgR), another Fc receptor for IgA and IgM. These results suggest that IgA and IgM bind to an epitope in the conserved amino acids in the Ig-like domain of Fcalpha/muR as well as poly-IgR.     

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.     

Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies

Background

Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety.

Methods

We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation.

Results

ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells.

Conclusion

A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.     

Ligand-free and -bound structures of the binding protein (LivJ) of the Escherichia coli ABC leucine/isoleucine/valine transport system: trajectory and dynamics of the interdomain rotation and ligand specificity

The leucine/isoleucine/valine-binding protein (LIVBP or LivJ) serves as the primary high-affinity receptor of the Escherichia coli ABC-type transporter for the three aliphatic amino acids. The first structure of LIVBP determined previously without bound ligand showed a molecule comprised of two domains which are far apart and bisected by a wide open, solvent-accessible cleft. Here we report the crystal structures of another ligand-free state and three complexes with the aliphatic amino acids. In the present ligand-free structure, the two domains are farther apart. In the three very similar complex structures, the two domains are in close proximity, and each desolvated ligand is completely engulfed in the cleft and bound by both domains. The two different ligand-free structures, combined with those of the very similar ligand-bound structures, indicate the trajectory and backbone torsion angle changes of the hinges that accompany domain closure and play crucial functional roles. The amino acids are bound by polar and nonpolar interactions, occurring predominantly in one domain. Consistent with the protein specificity, the aliphatic side chains of the ligands lie in a hydrophobic pocket fully formed following domain or cleft closure. Comparison of the structures of LIVBP with several different binding proteins indicates no correlations between the magnitudes of the hinge-bending angles and the protein masses, the ligand sizes, or the number of segments connecting the two domains. Results of normal-mode analysis and molecular dynamics simulations are consistent with the trajectory and intrinsic flexibility of the interdomain hinges and the dominance of one domain in ligand binding in the open state.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

Functional domains of the granulocyte colony-stimulating factor receptor.

The granulocyte colony-stimulating factor (G-CSF) receptor has a composite structure consisting of an immunoglobulin(Ig)-like domain, a cytokine receptor-homologous (CRH) domain and three fibronectin type III (FNIII) domains in the extracellular region. Introduction of G-CSF receptor cDNA into IL-3-dependent murine myeloid cell line FDC-P1 and pro-B cell line BAF-B03, which normally do not respond to G-CSF, enabled them to proliferate in response to G-CSF. On the other hand, expression of the G-CSF receptor cDNA in the IL-2-dependent T cell line CTLL-2 did not enable it to grow in response to G-CSF, although G-CSF could transiently stimulate DNA synthesis. Mutational analyses of the G-CSF receptor in FDC-P1 cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a portion of the cytoplasmic domain of about 100 amino acids in length were indispensable for transduction of the G-CSF-triggered growth signal.