Neutralizing antibodies against therapeutic proteins can be potentially harmful if the antibody blocks not only the therapeutic activities of the therapeutic protein but also the normal functions of the endogenous counterpart. Detection of the neutralizing anti-therapeutic protein antibodies generally relies on bioassays measuring changes in the biologic activity of the therapeutic protein triggered by the presence of the antibody. Most of the bioassays, particularly the cell-based in vitro assays, fail to detect neutralizing anti-therapeutic protein antibodies when the remaining therapeutic protein level in the assay samples is high. The remaining therapeutic protein, either a free molecule or an immune complex with anti-therapeutic protein antibodies, can inhibit the neutralizing activity of the antibody and prevent detection. We describe the development of a procedure that uses acid dissociation and affinity adsorption to remove therapeutic protein from assay samples. With this procedure, we can detect the presence of neutralizing anti-therapeutic protein antibodies from samples containing high levels of therapeutic protein.

The glial cell line-derived neurotrophic factor (GDNF) family coreceptor α1 (GFRα1) is a critical component of the RET receptor kinase signal-transducing complex. The activity of this multicomponent receptor is stimulated by the glial cell line-derived neurotrophic factor (GDNF) and is involved in neuronal cells survival and kidney development. GFRα1 pre-mRNA is alternatively spliced and produces two isoforms: GFRα1a, which includes the exon 5; and GFRα1b, which excludes it. Here we show that the Gfrα1a isoform is predominantly expressed in neuronal tissues and in PC12 cells differentiated toward a neuronal phenotype. GFRα1 splicing is also regulated during kidney development, GFRα1a is the minor isoform before birth and then rapidly becomes the major form after birth. We established cell lines expressing either GFRα1 isoforms and demonstrated that the GFRα1b isoform binds GDNF more efficiently than GFRα1a. Consistently, GFRα1b promotes a stronger RET phosphorylation than GFRα1a. These results indicate that specific inclusion of the GFRα1 exon 5 in neuronal tissues or during kidney development may alter the binding properties of GDNF to GFRα1, and thus could constitute an additional regulatory mechanism of the RET signaling pathway.

We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3(+) eosinophils increased in the blood and lymph nodes of the transgenic mice by 50- and 300-fold, respectively. Eosinophils also increased 8- to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19(+) B cells increased 2- to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4(+) T lymphocytes increased 2-fold in both blood and spleen. High serum levels of the cytokines IL-2, IL-4, IL-5, granulocyte colony-stimulating factor, eotaxin, and interferon gamma were observed. Consistent with B-lymphocyte increases, serum immunoglobulin (Ig) M, IgG, and IgE were significantly elevated. Antigenic challenge of the transgenic mice with keyhole limpet hemocyanin (KLH) resulted in a decrease in anti-KLH IgG accompanied by increases of anti-KLH IgA and IgE. In situ hybridization of transgenic tissues revealed that IL-17Rh1 (IL-17BR/Evi27), a receptor that binds IL-17E, is up-regulated. Taken together, these data indicate that IL-17E regulates hematopoietic and immune functions, stimulating the development of eosinophils and B lymphocytes. The fact that hIL-17E overexpression results in high levels of circulating eosinophils, IL-4, IL-5, eotaxin, and IgE suggests that IL-17E may be a proinflammatory cytokine favoring Th2-type immune responses.

The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been shown to play critical roles in cell migration, tissue morphogenesis, and axonal guidance in many different systems. However, their function in the spinal cord has not been examined carefully. We showed in this study that several Eph receptors, including EphA3, Eph A4, and Eph A5, are expressed in the ventral spinal cord in partially overlapping patterns, with EphA5 exhibiting the most widespread transcription in the entire ventral spinal cord during early development. Complementary to the receptor expression, a ligand of these receptors, ephrin-A5, is transcribed in the dorsal half of the spinal cord. Consistent with the spatial location of receptor expression, the ligand selectively inhibits neurite outgrowth and induces cell death of the ventral, but not the dorsal, spinal cord neurons. These observations suggest that interactions between the Eph family receptors and ligands exerts negative influences on ventral spinal cord neurons and thus may play important roles in regulating morphogenesis and axon guidance in the spinal cord.

Glial cell line-derived neurotrophic factor (GDNF) and a related factor, neurturin, promote survival of diverse groups of neurons. Both GDNF and neurturin signal via a two-component receptor complex that consists of a ligand-binding GDNF family receptor (GFRalpha-1 or GFRalpha-2) and the receptor protein tyrosine kinase Ret. Recently, a third receptor related to GFRalpha-1 and GFRalpha-2 has also been isolated and designated GFRalpha-3. Although much is known about the interaction among GDNF family factors, Ret, and the alpha-receptors in vitro, it remains unclear about their interactions in vivo. We show here by in situ hybridization that Ret and the alpha-receptors may be colocalized in the same tissues or expressed separately in projecting and target tissues, respectively, indicating that two distinct modes of interaction between Ret and the alpha-receptors exist in vivo. First, Ret may interact with the alpha-receptors expressed in the same cells (termed interaction "in cis") in many tissues and cell populations that respond to GDNF and/or neurturin, such as the substantia nigra, dorsal root ganglia, spinal cord motoneurons, kidney, and intestine. Second, Ret may interact with the alpha-receptors localized in the target neurons (termed interaction "in trans"). In addition, we present evidence in vitro that GFRalpha-1 mediates Ret activation by GDNF in trans. These observations suggest that there are multiple mechanisms regulating the interaction between Ret and the alpha-receptors that mediates the effects of GDNF family trophic factors on the survival and differentiation of cells and on neuron-target interactions in the nervous system.

Ret is a receptor tyrosine kinase involved in several neoplastic and developmental diseases affecting the thyroid gland and tissues of neuroectodermal origin. Different ret mutations are associated with different disease phenotypes. Gain-of-function of ret is caused by gene rearrangements in thyroid papillary carcinomas and by point mutations in multiple endocrine neoplasia (MEN) type 2A syndrome (MEN2A), in familial medullary thyroid carcinoma (FMTC), and in the more severe MEN2B syndrome. Conversely, Hirschsprung's disease (HSCR) is associated with loss of function of ret. Recently, it has been shown that glial cell line-derived neurotrophic factor (GDNF), by binding to the accessory molecule GDNFR-alpha, acts as a functional ligand of Ret and stimulates its tyrosine kinase and biological activity. To ascertain whether the biological effects of ret mutations are modulated by GDNF, we have investigated the responsiveness to GDNF of ret mutants in cell lines coexpressing GDNFR-alpha and MEN2A-, MEN2B-, FMTC-, or HSCR-associated ret mutants. Here, we show that triggering of GDNF affected only ret/MEN2B, i.e. it stimulated ret/MEN2B mitogenic and kinase activities, as well as its ability to phosphorylate Shc, a bona fide Ret substrate. In contrast, ret mutants associated with MEN2A or FMTC (carrying Cys634 or Cys620 mutations) were unresponsive to GDNF. HSCR mutations, by affecting either the extracellular or the intracellular Ret domain, impaired responsiveness to GDNF. These data suggest that the phenotype of human diseases caused by ret mutations can be differentially influenced by GDNF.

Members of the glial cell line–derived neurotrophic factor (GDNF) family, including GDNF and neurturin (NTN), play key roles in the control of vertebrate neuron survival and differentiation. GDNF and NTN signal via a multicomponent receptor system formed by a glycosyl-phosphatidylinositol (GPI)-linked ligand binding subunit (the “α” component) and the receptor tyrosine kinase RET as a signaling (i. e. “β”) subunit. To date, two distinct genes encoding α receptor components for GDNF and NTN have been reported in the literature. The first member of this receptor family, GDNF receptor-α (GDNFR-α) was shown to bind GDNF and to mediate binding and activation of the RET receptor tyrosine kinase (3Jing S.Q. Wen D.Z. Yu Y.B. Holst P.L. Luo Y. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar, 7Treanor J. Goodman L. Desauvage F. Stone D.M. Poulsen K.T. Beck C.D. Gray C. Armanini M.P. Pollock R.A. Hefti F. Phillips H.S. Goddard A. Moore M.W. Bujbello A. Davies A.M. Asai N. Takahashi M. Vandlen R. Henderson C.E. Rosenthal A. Nature. 1996; 382: 80-83Crossref PubMed Scopus (943) Google Scholar). The second member has been shown to bind NTN and to mediate activation of RET by both NTN and GDNF (1Baloh R.H. Tansey M.G. Golden J.P. Creedon D.J. Heuckeroth R.O. Keck C.L. Zimonjic D.B. Popescu N.C. Johnson E.M. Milbrandt J. Neuron. 1997; 18: 793-802Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 2Bujbello A. Adu J. Pinon L. Horton A. Thompson J. Rosenthal A. Chinchetru M. Buchman V.L. Davies A.M. Nature. 1997; 387: 721-724Crossref PubMed Scopus (253) Google Scholar, 4Klein R.D. Sherman D. Ho W.H. Stone D. Bennett G.L. Moffat B. Vandlen R. Simmons L. Gu Q.M. Hongo J.A. Devaux B. Poulsen K. Armanini M. Nozaki C. Asai N. Goddard A. Phillips H. Henderson C.E. Takahashi M. Rosenthal A. Nature. 1997; 387: 717-721Crossref PubMed Scopus (340) Google Scholar, 5Sanicola M. Hession C. Worley D. Carmillo P. Ehrenfels C. Walus L. Robinson S. Jaworski G. Wei H. Tizard R. Whitty A. Pepinsky R.B. Cate R.L. Proc. Natl. Acad. Sci. USA. 1997; 94: 6238-6243Crossref PubMed Scopus (272) Google Scholar, 6Suvanto P. Wartiovaara K. Lindahl M. Arumäe U. Moshnyakov M. Horelli-Kuitunen N. Airaksinen M.S. Palotie A. Sariola H. Saarma M. Hum. Mol. Gen. 1997; 6: 1267-1273Crossref PubMed Scopus (81) Google Scholar). This protein has been alternatively named TrnR-2, NTNR-α, RETL2, and GDNFR-β. Additional members of the GDNF ligand family as well as α receptor components are known to exist. In an effort to arrive at a sensible and easy to use consensus nomenclature for the α receptor components for the GDNF ligand family, several of us who have been involved in the identification and characterization of these receptors have formed a committee with the objective of proposing a nomenclature for the members of this receptor family. We propose to use the abbreviated name “GFRα-X” (for “GDNF Family Receptor Alpha-X”) where “X” denotes an arabic numeral to be assigned based on the date of publication of the receptor. Thus, GDNFR-α will become GFRα-1, while NTNR-α/TrnR-2/RETL2/GDNFR-β will be GFRα-2. The root name for the corresponding gene will be GFRα-X. We feel that this nomenclature is flexible enough to allow for the possibility of cross-talk between ligands and receptors. In addition, the use of the term “alpha” will serve to clarify the fact that these molecules are part of a multicomponent receptor complex and are all structurally related. We strongly encourage all investigators of GDNF family ligands and receptors to adopt these nomenclature recommendations in order to achieve our goal of improving communication between researchers both inside and outside this exciting field.1The following scientists have endorsed this nomenclature: Alun M. Davies, Jack E. Dixon, Gary M. Fox, Carlos F. Ibáñez* (chair), Shuqian Jing, Eugene Johnson, Jeffrey Milbrandt, Heidi Phillips, Arnon Rosenthal, Mart Saarma, Michele Sanicola, James Treanor, and Quinn C. Vega. *Laboratory of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden ([email protected])*Laboratory of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden ([email protected])

The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFRalpha-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFRalpha-1 or GFRalpha-2, a novel receptor related to GFRalpha-1. Both GFRalpha-1 and GFRalpha-2 mediate neurturin-induced Ret phosphorylation. GDNF can also bind to either GFRalpha-1 or GFRalpha-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFRalpha-1 bind GDNF more efficiently than neurturin, while cells expressing GFRalpha-2 bind neurturin preferentially. Cross-linking and Ret activation data also suggest that while there is cross-talk, GFRalpha-1 is the primary receptor for GDNF and GFRalpha-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFRalpha receptor family. This putative receptor, designated GFRalpha-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFRalpha-1 and GFRalpha-2. Analysis of the tissue distribution of GFRalpha-1, GFRalpha-2, GFRalpha-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFRalphas and Ret are expressed in many of the same tissues, suggesting that GFRalphas mediate the action of GDNF family ligands in vivo.

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.