Fibroblast growth factors (FGFs) constitute a large family of at least nine distinct polypeptide growth factors (7,31,34). FGFs play an important role in the regulation of cell growth, differentiation, embryogenesis, and angiogenesis (34). Like other growth factors, FGFs exert their action by binding to and activating a distinct family of growth factor receptors that has been previously classified as subclass IV (20, 74). The FGF receptor family consists of at least four distinct gene products, each composed of an extracellular ligand-binding domain that contains three immunoglobulin-like domains, a single transmembrane domain, and a cytoplasmic domain that contains protein tyrosine kinase activity (interrupted by an insertion of 14 amino acids in the kinase domain). One of the characteristic features of the FGF receptor family is the occurrence of numerous receptor isoforms that are produced from alternatively spliced transcripts in both the intracellular and extracellular domains (9,14,27,32,33). As with other growth factors, binding of FGF to FGF receptors leads to receptor dimerization (3, 70, 73) and subsequent tyrosine autophosphorylation and phosphorylation of target substrates (6, 13, 23). Autophosphorylation on tyrosine is considered to have at least two functions. One such function is the stimulation of the intrinsic protein tyrosine kinase activity by an allosteric mechanism, as seen with the insulin receptor (22,63,81,84,86). Also, many autophosphorylation sites serve as binding sites for signaling proteins that contain Src homology 2 (SH2) domains or the recently identified phosphotyrosine interaction (also called phosphotyrosine-binding [PTB]) domains (4,5,8,35,36,39,48,56,57). Binding of SH2 or PTB domain-containing proteins to activated growth factor receptors has been shown to be important for the activation of downstream signaling molecules. For example, binding of Shc and phospholipase C␥ (PLC␥) through PTB and SH2 domains, respectively, to the activated nerve growth factor receptor (Trk) has been shown to be required for the nerve growth factor-induced activation of Ras signaling pathways and neuronal differentiation of PC12 cells (16,55,72). Several studies have demonstrated that mutation of autophosphorylation sites in platelet-derived growth factor (PDGF) receptor and in colony-stimulating factor 1 receptor can impair mitogenic signaling in some cell lines (19,76,77).Very little is known about the cellular substrates and target proteins involved in signaling processes that lead to FGFmediated mitogenesis. So far only PLC␥ has been shown to associate with the activated FGF receptor 1 (FGFR1) (flg); the identities of other targets remain unclear. We have previously identified tyrosine 766 of FGFR1 as the major autophosphorylation site and have shown that this tyrosine and its flanking sequences represent a high affinity binding site for one of the SH2 domains of PLC␥ (52). Mutation of this tyrosine to phenylalanine results in a receptor that is no longer able to stimulate phosphatidylinosito...

Pyk2 is a protein tyrosine kinase that links G-proteincoupled receptors, inflammatory cytokines, and extracellular stimuli that elevate intracellular calcium concentration with activation of the mitogen-activated protein kinase pathways and regulation of ion channel functions. Here we describe the identification, cloning, and characterization of a new isoform of Pyk2 (Pyk2-H) that is generated by alternative RNA splicing. Pyk2-H is mainly expressed in hematopoietic cells including Tcells, B-cells, and natural killer cells. Engagement of T-cell or B-cell antigen receptors leads to rapid tyrosine phosphorylation of Pyk2-H. Pyk2-H is also activated in response to the chemokines RANTES and macrophage inflammatory protein-1␤ in T cells. In addition, we show that glutathione S-transferase fusion proteins containing the carboxyl termini of Pyk2 and Pyk2-H bind to a different set of tyrosine-phosphorylated proteins in thymus lysates. Specific expression of Pyk2-H and its activation by antigens or chemokines in hematopoietic cells may contribute toward the generation of cell type-specific signals involved in host immune responses.Pyk2 together with focal adhesion kinase (FAK) 1 constitute a distinct family of non-receptor protein tyrosine kinases that are regulated by extracellular stimuli (1, 2). Pyk2 is predominantly expressed in the central nervous system and in cells and tissues derived from hematopoietic lineages (2-4). In neuronal cells, Pyk2 links various extracellular stimuli that lead to elevation of intracellular calcium concentration, such as activation of the G-protein-coupled receptors, nicotinic acetylcholine receptors, and membrane depolarization, with ion channel and mitogen-activated protein kinase functions (2, 5). Furthermore, we have shown that Pyk2 acts in concert with the Src protein tyrosine kinase to activate the mitogen-activated protein kinase signaling pathway in response to activation of G i and G q coupled receptors in PC12 cells (5). Pyk2 was implicated as an upstream regulator of the JNK/SAPK signaling pathway in response to stress stimuli, inflammatory cytokines, or activation of angiotensin II receptors (6, 7). It has also been demonstrated that Pyk2 plays a role in signaling by lymphocyte antigen receptors, integrin receptors, and chemokine receptors in hematopoietic cells (9 -12).We and others have observed that in some cells of hematopoietic origin Pyk2 protein migrates in SDS-PAGE gels as a doublet (9, 11). This observation suggested that different Pyk2 isoforms may be expressed in hematopoietic cells. In this report we describe the identification, cloning, and initial characterization of an isoform of Pyk2, that is generated by alternative RNA splicing, and is mainly expressed in hematopoietic cells (Pyk2-H). Using isoform-specific anti-Pyk2 antibodies we show that Pyk2-H is expressed in primary T and B lymphocytes and in natural killer cells. We demonstrate that Pyk2-H and not Pyk2 is rapidly activated in response to stimulation with chemokines and following activation of the T-ce...