RESUMEN
The ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaigns has direct impact on the timelines of a project. Automated liquid handling (ALH)-based mAb purification platforms have been used to facilitate the production of large numbers of mAbs. However, the ongoing pressure to de-risk potential lead molecules at an early development stage by including bio-physical characterization of mAbs has further increased the demand to produce sufficient quantities from limited sample volumes. A bottleneck so far has been the limited dynamic binding capacity of these systems, which is partly due to the binding properties of commonly used Protein A affinity matrices. The present publication suggests that by using a Protein A matrix optimized for continuous chromatography applications the yields of ALH-based but also standard lab-scale mAb purifications can be significantly increased without the need to change established protocols.
Asunto(s)
Anticuerpos Monoclonales/química , Proteínas Recombinantes de Fusión/química , Anticuerpos Monoclonales/genética , Células Cultivadas , Cromatografía de Afinidad , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Recombinantes de Fusión/genética , Robótica , Proteína Estafilocócica A/química , TransfecciónRESUMEN
We describe a novel cloning method, referred to as insert-tagged (InTag) positive selection, for the rapid one-step reformatting of phage-displayed antibody fragments to full-length immunoglobulin Gs (IgGs). InTag positive selection enables recombinant clones of interest to be directly selected without cloning background, bypassing the laborious process of plating out cultures and colony screening and enabling the cloning procedure to be automated and performed in a high-throughput format. This removes a significant bottleneck in the functional screening of phage-derived antibody candidates and enables a large number of clones to be directly reformatted into IgG without the intermediate step of Escherichia coli expression and testing of soluble antibody fragments. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated, and optimized methods for the small-scale transient expression of IgGs at high levels are described. InTag positive selection cloning has the potential for wide application in high-throughput DNA cloning involving multiple inserts, markedly improving the speed and quality of selections from protein libraries.
Asunto(s)
Técnicas de Visualización de Superficie Celular , Inmunoglobulina G/genética , Fragmentos de Inmunoglobulinas/genética , Inmunoglobulina G/biosíntesis , TransfecciónRESUMEN
Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Epítopos , Subunidad alfa1 del Receptor de Interleucina-13/química , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Sitios de Unión/inmunología , Cristalografía por Rayos X , Dimerización , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Interleucina-13/inmunología , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Leucina/metabolismo , Ratones , Ratones Transgénicos , Estructura Terciaria de ProteínaRESUMEN
IL-11 and IL-11 receptor (R)alpha are induced by Th2 cytokines. However, the role(s) of endogenous IL-11 in antigen-induced Th2 inflammation has not been fully defined. We hypothesized that IL-11, signaling via IL-11Ralpha, plays an important role in aeroallergen-induced Th2 inflammation and mucus metaplasia. To test this hypothesis, we compared the responses induced by the aeroallergen ovalbumin (OVA) in wild-type (WT) and IL-11Ralpha-null mutant mice. We also generated and defined the effects of an antagonistic IL-11 mutein on pulmonary Th2 responses. Increased levels of IgE, eosinophilic tissue and bronchoalveolar lavage (BAL) inflammation, IL-13 production, and increased mucus production and secretion were noted in OVA-sensitized and -challenged WT mice. These responses were at least partially IL-11 dependent because each was decreased in mice with null mutations of IL-11Ralpha. Importantly, the administration of the IL-11 mutein to OVA-sensitized mice before aerosol antigen challenge also caused a significant decrease in OVA-induced inflammation, mucus responses, and IL-13 production. Intraperitoneal administration of the mutein to lung-specific IL-13-overexpressing transgenic mice also reduced BAL inflammation and airway mucus elaboration. These studies demonstrate that endogenous IL-11R signaling plays an important role in antigen-induced sensitization, eosinophilic inflammation, and airway mucus production. They also demonstrate that Th2 and IL-13 responses can be regulated by interventions that manipulate IL-11 signaling in the murine lung.
Asunto(s)
Inflamación/metabolismo , Interleucina-11/metabolismo , Interleucina-13/metabolismo , Moco/metabolismo , Transducción de Señal , Células Th2/metabolismo , Alérgenos/inmunología , Animales , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Regulación de la Expresión Génica/efectos de los fármacos , Inmunización , Ratones , Ratones Endogámicos C57BL , Mucina 5AC/genética , Mucina 5AC/metabolismo , Ovalbúmina/inmunología , Fenotipo , Receptores de Interleucina-11/metabolismo , Transducción de Señal/efectos de los fármacos , Células Th2/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Mice deficient in SOCS2 display an excessive growth phenotype characterized by a 30-50% increase in mature body size. Here we show that the SOCS2-/- phenotype is dependent upon the presence of endogenous growth hormone (GH) and that treatment with exogenous GH induced excessive growth in mice lacking both endogenous GH and SOCS2. This was reflected in terms of overall body weight, body and bone lengths, and the weight of internal organs and tissues. A heightened response to GH was also measured by examining GH-responsive genes expressed in the liver after exogenous GH administration. To further understand the link between SOCS2 and the GH-signaling cascade, we investigated the nature of these interactions using structure/function and biochemical interaction studies. Analysis of the 3 structural motifs of the SOCS2 molecule revealed that each plays a crucial role in SOCS2 function, with the conserved SOCS-box motif being essential for all inhibitory function. SOCS2 was found to bind 2 phosphorylated tyrosines on the GH receptor, and mutational analysis of these amino acids showed that both were essential for SOCS2 function. Together, the data provide clear evidence that SOCS2 is a negative regulator of GH signaling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hormona del Crecimiento/fisiología , Receptores de Somatotropina/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Secuencias de Aminoácidos/genética , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Peso Corporal/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Hormona del Crecimiento/administración & dosificación , Hormona del Crecimiento/genética , Factor I del Crecimiento Similar a la Insulina/fisiología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Fosforilación , Unión Proteica/genética , Unión Proteica/fisiología , Receptores de Somatotropina/genética , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/genética , Tirosina/metabolismoRESUMEN
Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Robótica , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Automatización , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Células HEK293 , Humanos , Miniaturización , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismoRESUMEN
The ß common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared ß common (ßc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human ßc receptor. The binding epitope of CSL311 on the ßc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human ßc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 ß common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human ßc receptor is central to pathogenesis. The coordinates for the ßc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).
Asunto(s)
Anticuerpos Monoclonales de Origen Murino , Subunidad beta Común de los Receptores de Citocinas , Epítopos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Interleucina-3 , Interleucina-5 , Animales , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Asma/tratamiento farmacológico , Asma/inmunología , Asma/patología , Cristalografía por Rayos X , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/inmunología , Eosinófilos/inmunología , Eosinófilos/patología , Epítopos/química , Epítopos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Interleucina-3/antagonistas & inhibidores , Interleucina-3/inmunología , Interleucina-5/antagonistas & inhibidores , Interleucina-5/inmunología , RatonesRESUMEN
SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase-signal transduction and activator of transcription (JAK-STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N-terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C-terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C-terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single alpha-helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding.
Asunto(s)
Estructura Secundaria de Proteína , Proteínas Supresoras de la Señalización de Citocinas/química , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Clonación Molecular , Humanos , Ratones , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fosfotirosina/metabolismo , Alineación de Secuencia , Transducción de Señal/fisiología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Subunidad alfa del Receptor de Interleucina-3/química , Secuencia de Aminoácidos , Cristalización , Humanos , Datos de Secuencia Molecular , Unión Proteica , Difracción de Rayos XRESUMEN
The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.
Asunto(s)
Astrocitos/inmunología , Axones/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Receptor EphA4/genética , Médula Espinal/inmunología , Traslado Adoptivo , Animales , Astrocitos/patología , Axones/patología , Movimiento Celular , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Femenino , Eliminación de Gen , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/farmacología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/farmacología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Receptor EphA4/antagonistas & inhibidores , Receptor EphA4/inmunología , Índice de Severidad de la Enfermedad , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Linfocitos T/trasplanteRESUMEN
Vascular endothelial growth factor (VEGF) B effects blood vessel formation by binding to VEGF receptor 1. To study the specifics of the biological profile of VEGF-B in both physiological and pathological angiogenesis, a neutralising anti-VEGF-B antibody (2H10) that functions by inhibiting the binding of VEGF-B to VEGF receptor 1 was developed. Here, we present the structural features of the 'highly ordered' interaction of the Fab fragment of this antibody (Fab-2H10) with VEGF-B. Two molecules of Fab-2H10 bind to symmetrical binding sites located at each pole of the VEGF-B homodimer, giving a unique U-shaped topology to the complex that has not been previously observed in the VEGF family. VEGF-B residues essential for binding to the antibody are contributed by both monomers of the cytokine. Our detailed analysis reveals that the neutralising effect of the antibody occurs by virtue of the steric hindrance of the receptor-binding interface. These findings suggest that functional complementarity between VEGF-B and 2H10 can be harnessed both in analysing the therapeutic potential of VEGF-B and as an antagonist of receptor activation.
Asunto(s)
Anticuerpos/química , Fragmentos Fab de Inmunoglobulinas/química , Factor B de Crecimiento Endotelial Vascular/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Estructura Secundaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclized backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Taking advantage of this stable framework we developed novel VEGF-A antagonists by grafting a peptide epitope involved in VEGF-A antagonism onto the stable cyclotide framework. Antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. A grafted analogue showed biological activity in an in vitro VEGF-A antagonism assay at low micromolar concentration and the in vitro stability of the target epitope was markedly increased using this approach. In general, the stabilization of bioactive peptide epitopes is a significant problem in medicinal chemistry and in the current study we have provided insight into one approach to stabilize these peptides in a biological environment.
Asunto(s)
Ciclotidas/química , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Química Farmacéutica/métodos , Motivos Nodales de Cisteina , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Epítopos/química , Hemólisis , Humanos , Concentración de Iones de Hidrógeno , Modelos Químicos , Conformación Molecular , Neovascularización Patológica , Estructura Terciaria de ProteínaRESUMEN
Suppressor of cytokine signaling-3 (SOCS-3) and the protein tyrosine phosphatase SHP-2 both regulate signaling by cytokines of the interleukin-6 family, and this is dependent upon recruitment to tyrosine 757 in the shared cytokine receptor subunit gp130. To better explore the overlap in ligand binding specificities exhibited by these two signaling regulators, we have mapped the phosphopeptide binding preferences of the SH2 domains from SOCS-3 and SHP-2. Degenerate phosphopeptide libraries were screened against recombinantly produced SH2 domains to determine the sequences of optimal phosphopeptide ligands. We found that the consensus ligand binding motif for SOCS-3 was pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic-(H/V/I/Y), while the consensus motif for SHP-2 was pY-(S/T/A/V/I)-X-(V/I/L)-X-(W/F). We validated these data through the design of phosphopeptide ligands based on the consensus motifs and found that these bound to SOCS-3 and SHP-2 with high affinity. Finally, we have compared the affinity of SOCS-3 for binding to phosphopeptides representing putative docking sites in the gp130, leptin and erythropoietin receptors. While SOCS-3 binds with much higher affinity to a gp130 phosphopeptide than to phosphopeptides derived from the other receptors, multiple SOCS-3 binding sites are predicted to exist in the leptin and erythropoietin receptors which may compensate for weaker binding to individual sites.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismo , Proteínas Represoras , Factores de Transcripción , Dominios Homologos src , Péptidos y Proteínas de Señalización Intracelular , Leptina/metabolismo , Ligandos , Fosfopéptidos/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas/química , Receptores de Eritropoyetina/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Especificidad por Sustrato , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de CitocinasRESUMEN
Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.