These results indicate that FGF-8 is an AER-derived mitogen that stimulates limb bud outgrowth. Moreover, our data suggest that FGF-8 may also be an ectodermally derived mitogen that stimulates the onset of limb bud outgrowth (budding) in the absence of a morphological AER, and indicate the possible involvement of FGF-8 in the establishment of the limb field.

fgf3 has been implicated in the embryonic and fetal development of the mouse and as an oncogene in murine breast cancer. We describe a procedure to purify the product of the mouse fgf3 gene and show it to be a potent mitogen for some epithelial cell lines. Using a receptor binding competition assay, Fgf3 was shown to bind with high affinity to the IIIb isoforms of Fgf receptor (FgfR) 1 and FgfR2 (ID 50 The fibroblast growth factors constitute a family of nine proteins that share 35-55% amino acid identity over a core region (Refs. 1 and 2; reviewed in Refs. 3 and 4). The prototypic members, Fgf1 and Fgf2, have been ascribed a number of properties including the induction of cell proliferation, differentiation, migration, and cell survival, consistent with roles as autocrine and paracrine signaling molecules. For Fgf2 and Fgf3 there is also good evidence for the translocation of the protein directly to the cell nucleus (5-8); however, the biological significance of this event is not known. The common route for Fgf signaling is through an interaction of an extracellular Fgf with cell surface receptors (reviewed in Refs. 9 and 10). Two classes of Fgf receptor have been identified: a low affinity receptor typified by heparan sulfate proteoglycans that bind Fgfs to high capacity but seem not to signal (11) and a high affinity receptor with intrinsic tyrosine kinase activity (reviewed in Refs. 9 and 10). Four tyrosine kinase receptor genes (fgfr1 to fgfr4) have been identified in mammals that encode an extracellular ligand binding domain composed of two (␤-form) or three (␣-form) immunoglobulin-like motifs, a transmembrane segment, and a cytoplasmic portion that encompasses a tyrosine kinase domain. FgfR1, 1 FgfR2, and FgfR3 but not FgfR4 have a choice of exon encoding the second half of the third Ig loop, termed IIIb and IIIc, respectively. This alternative splicing changes the ligand binding specificity of the receptors (12-15). Both receptor classes are needed for signal transduction. The low affinity receptors are required for high affinity ligand binding and appear to facilitate the dimerization of the tyrosine kinase receptor-Fgf complexes (16 -18). Dimer formation results in tyrosine autophosphorylation of the receptor providing suitable sites for second messenger interactions and consequent signal transduction (reviewed in Ref. 19).fgf3 was identified as a proto-oncogene activated by proviral insertion in mouse mammary tumors (20,21). Expression of fgf3 was not detected in the normal mammary glands, suggesting that its inappropriate expression contributed to tumorigenesis. This notion gained considerable support from transgenic mouse studies (22-24). Thus, constitutive ectopic expression of fgf3 led to abnormal mammary gland development manifest as multifocal pregnancy-sensitive epithelial hyperplasia, with the stochastic appearance of frank neoplasia. These observations prompted us to identify the receptors responsible for Fgf3 signaling and to determine which isoforms are present on mammary epithelial cells....

The superfamily of GPCRs have diverse biological roles, transducing signals from a range of stimuli, from photon recognition by opsins to neurotransmitter regulation of neuronal function. Of the many identified genes encoding GPCRs, >130 are orphan receptors ( i.e., their endogenous ligands are unknown), and this subset represents putative novel therapeutic targets for pharmaceutical intervention in a variety of diseases. As an initial step toward drug discovery, determining a biological function for these newly identified receptors is of vital importance, and thus identification of a natural ligand(s) is a primary aim. There are several established methods for doing this, but many have drawbacks and usually require some in-depth knowledge about how the receptor functions. The technique described here utilizes a transcription-based reporter assay in live cells. This allows the determination of the signal transduction pathway any given oGPCR uses, without any prior knowledge of the endogenous ligand. This can therefore reduce the redundancy of effort involved in screening ligands at a given receptor in multiple formats (i.e., Galpha(s), Galpha(i/0), and Galpha(q) assays), as well as ensuring that the receptor targeted is capable of signaling if appropriately activated. Such knowledge is often laboriously obtained, and for almost all oGPCRs, this kind of information is not yet available. This technology can also be used to develop inverse agonist as well as agonist sensitive high throughput assays for oGPCRs. The veracity of this approach is demonstrated, using a number of known GPCRs. The likely signaling pathways of the GPR3, GPR12, GPR19, GPR21, and HG55 oGPCRs are shown, and a high throughput assay for GPR26 receptors developed. The methods outlined here for elucidation of the signal transduction pathways for oGPCRs and development of functional assays should speed up the process of identification of ligands for this potentially therapeutically useful group of receptors.

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