MOLECULAR CLONING OF THE GUINEA-PIG IL-5 RECEPTOR Α AND Β SUBUNITS AND RECONSTITUTION OF a HIGH AFFINITY RECEPTOR

Scott, Clay W.; Logsdon, Naomi J.; Wilkins, Deidre; Norris, Tyrrell E.; Sobotka-Briner, Cindy; Hubbs, Stephen J.; Graham, Alexander

doi:10.1006/cyto.1999.0657

Cited by 3 publications

(5 citation statements)

References 37 publications

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“…2). 18 Analysis of equilibrium binding data indicates a single population of saturable binding sites with a K d =220 pM. This affinity is consistent with that shown for the binding of hIL-5 to human eosinophils.…”

Section: Cloning and Expression Of Guinea Pig Il-5supporting

confidence: 84%

“…Recombinant gpIL-5rab 1 and hIL-5rab complexes were produced in Sf9 cells as previously described. 18 Recombinant hIL-5 was expressed in Sf9 cells using procedures described by Brown e t a l. 19 IL-5 purification hIL-5 was purified by a combination of Q-Sepaharose, hydoxylapatite and phenyl Sepaharose column chromatography as described by Johanson e t a l. 20 gpIL-5 was purified in a single step by negative selection ion exchange chromatography on Q-Sepharose ff 19 equilibrated with 10 mM Tris-HCl, pH 8. The gpIL-5 eluted in the exclusion volume while the remaining proteins were bound to the column.…”

Section: Expressing Recombinant Proteins Using Baculovirus Expressionmentioning

confidence: 99%

“…Insect cells expressing either hIL-5rab or gpIL-5rab 1 were processed for binding assays as previously described. 18 Plasma membrane preparations (5 mg aliquots) were incubated with 50 pM [ 125 I]hIL-5 in assay buffer consisting of 25 mM Tris, pH 7.4 and 150 mM NaCl. Some samples also received various concentrations of unlabeled human, guinea pig, or murine IL-5.…”

Section: Binding Assaymentioning

confidence: 99%

“…25 -27 A similar high affinity binding site is observed with [ 125 I]hIL-5 and membrane preparations expressing guinea pig IL-5r a and b 1 subunits (K d =110 pM). 18 These two preparations were used in displacement binding assays to determine whether gpIL-5 binds to gpIL-5rab 1 with an affinity similar to that seen with the human and murine ligand/receptor pairs and also to determine the relative affinities of guinea pig, murine and human IL-5 for the guinea pig and human IL-5 receptors.…”

Section: Cloning and Expression Of Guinea Pig Il-5mentioning

confidence: 99%

“…11,17 We recently reported the cloning and expression of guinea pig IL-5 receptor a subunit and multiple b subunits. 18 We also reconstituted a high affinity gpIL-5rab 1 complex using hIL-5 as ligand. In this report, we extend these studies to show the high affinity interaction of guinea pig IL-5 with its recombinant receptor.…”

Section: Introductionmentioning

confidence: 99%

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Expression and in vitro properties of guinea pig IL‐5: Comparison to human and murine orthologs

Scott

Budzilowicz

Hubbs

et al. 2000

Mediators of Inflammation

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Interleukin-5 (IL-5) is a key mediator of eosinophilic inflammation. The biological role of this cytokine in an allergic airway inflammatory response has been widely demonstrated in guinea pigs, yet the interaction of guinea pig IL-5 (gpIL-5) with its receptor has not been studied. Experiments were performed to quantitate the interaction of gpIL-5 with gpIL-5r and to compare this affinity with that of hIL-5 and mIL-5 and their cognate receptors. The cross-species affinity and agonist efficacy were evaluated to see if gpIL-5r had a restricted species reactivity (as is the case with mIL-5r) or did not distinguish between IL-5 orthologs (similar to hIL-5r). gpIL-5 was cloned using mRNA isolated from cells obtained by bronchoalveolar lavage. Recombinant gpIL-5 was expressed in T. ni insect cells and purified from spent media. Binding assays were performed using insect cells expressing hIL-5ralphabeta or gpIL-5ralphabeta1 as previously described (Cytokine, 12:858-866, 2000) or using B13 cells which express mIL-5r. The agonist potency and efficacy properties of each IL-5 ortholog were evaluated by quantitating the proliferative response of human TF-1 cells and murine B13 cells. gpIL-5 bound with high affinity to recombinant gpIL-5r as demonstrated by displacing [125I]hIL-5 (Ki = 160 pM). gpIL-5 also bound to hIL-5r with high affinity (Ki = 750 pM). hIL-5 and mIL-5 showed similar, high-affinity binding profiles to both gpIL-5r and hIL-5r. In contrast, gpIL-5 and hIL-5 did not bind to the mIL-5r as demonstrated by an inability to displace [125I]mIL-5, even at 1000-fold molar excess. These differences in affinity for IL-5r orthologs correlated with bioassay results: human TF-1 cells showed roughly comparable proliferative responses to guinea pig, human and murine IL-5 whereas murine B13 cells showed a strong preference for murine over guinea pig and human IL-5 (EC50 = 1.9, 2200 and 720 pM, respectively). Recombinant gpIL-5 binds to the gpIL-5r with high affinity, similar to that seen with the human ligand-receptor pair. gpIL-5r and hIL-5r do not distinguish between the three IL-5 orthologs whereas mIL-5r has restricted specificity for its cognate ligand.

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Section: Cloning and Expression Of Guinea Pig Il-5supporting

confidence: 84%

Section: Expressing Recombinant Proteins Using Baculovirus Expressionmentioning

confidence: 99%

Section: Binding Assaymentioning

confidence: 99%

Section: Cloning and Expression Of Guinea Pig Il-5mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Expression and in vitro properties of guinea pig IL‐5: Comparison to human and murine orthologs

Scott

Budzilowicz

Hubbs

et al. 2000

Mediators of Inflammation

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Molecular cloning of chicken interleukin-5 receptor α-chain and analysis of its binding specificity

Fukushima¹,

Miyai²,

Kumagae³

et al. 2012

Developmental & Comparative Immunology

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Using guinea pigs in studies relevant to asthma and COPD

Canning

Chou

2008

Pulmonary Pharmacology & Therapeutics

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The guinea pig has been the most commonly used small animal species in preclinical studies related to asthma and COPD. The primary advantages of the guinea pig are the similar potencies and efficacies of agonists and antagonists in human and guinea pig airways and the many similarities in physiological processes, especially airway autonomic control and the response to allergen. The primary disadvantages to using guinea pigs are the lack of transgenic methods, limited numbers of guinea pig strains for comparative studies and a prominent axon reflex that is unlikely to be present in human airways. These attributes and various models developed in guinea pigs are discussed.

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MOLECULAR CLONING OF THE GUINEA-PIG IL-5 RECEPTOR Α AND Β SUBUNITS AND RECONSTITUTION OF a HIGH AFFINITY RECEPTOR

Cited by 3 publications

References 37 publications

Expression and in vitro properties of guinea pig IL‐5: Comparison to human and murine orthologs

Expression and in vitro properties of guinea pig IL‐5: Comparison to human and murine orthologs

Molecular cloning of chicken interleukin-5 receptor α-chain and analysis of its binding specificity

Using guinea pigs in studies relevant to asthma and COPD

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