Saturation Mutagenesis of the WSXWS Motif of the Erythropoietin Receptor

Hilton, Douglas J.; Watowich, Stephanie S.; Katz, Luba; Lodish, Harvey F.

doi:10.1074/jbc.271.9.4699

Cited by 94 publications

(33 citation statements)

References 60 publications

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“…This domain of the receptor is known as the dimerization platform in the extracellular region of the receptor. WS motifs (215–219) in the D2 domain are required for the correct PRLR folding [17, 18]. It is interesting to note that mutations of two residues (Gln 187 and Glu 181) at the D2/D2 interface previously predicted to form H-bonding between the D2 domains of the S1b homodimers [10] did not alter the inhibitory function in the present study.…”

Section: Discussionmentioning

confidence: 62%

Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin

Kang

Hassan

Zhao

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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BACKGROUND Long-form (LF) homodimers of the human prolactin receptor (PRLR) mediate prolactin’s diverse actions. Short form S1b inhibits the LF function through heterodimerization. Reduced S1b/LF-ratio in breast cancer could contribute to tumor development/progression. Current work defines the structural and functional relevance of the D1 domain of S1b on its inhibitory function on prolactin-induced LF function. METHODS Mutagenesis, promoter/signaling analyses, bioluminescence resonance energy transfer (BRET), molecular modeling approaches. RESULTS Mutation of E69 in D1 S1b or adjacent residues at the receptor surface near to the binding pocket (S) causes loss of its inhibitory effect while mutations away from this region (A) or in the D2 domain display inhibitory action as the wild-type. All S1b mutants preserved prolactin-induced Jak2 activation. BRET reveals increased affinity in D1 mutated S1b (S) homodimers in transfected cells stably expressing LF. In contrast, affinity in S1b homodimers with either D1 (A) or D2 mutations remained unchanged. This favors LF mediated signaling induced by prolactin. Molecular dynamics simulations show that mutations (S) elicit major conformational changes that propagate downward to the D1/D2 interface and change their relative orientation in the dimers. CONCLUSIONS These findings demonstrate the essential role of D1 on the S1b structure and its inhibitory action on prolactin-induced LF-mediated function. GENERAL SIGNIFICANCE Major changes in receptor conformation and dimerization affinity are triggered by single mutations in critical regions of D1. Our structure-function/simulation studies provide a basis for modeling and design of small molecules to enhance inhibition of LF activation for potential use in breast cancer treatment.

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Section: Discussionmentioning

confidence: 62%

Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin

Kang

Hassan

Zhao

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…Mutants B13 and B15 contain proline rich regions that may be important for protein folding or loop stability. Mutant 21 contains the “WSXWS” motif (LSDWS in mEbi3), which is important for cytokine binding and may contribute to protein folding (Hilton et al, 1996). Mutants B16 and B20 contain several point mutations already shown to inhibit IL-27 dimer formation (F141A, K144A, D187A, D190A, Y191A), although they do not completely abrogate p28 binding in the context of the alanine scan mutants, suggesting other mutations may compensate somewhat for their negative effects.…”

Section: Resultsmentioning

confidence: 99%

Distinct subunit pairing criteria within the heterodimeric IL-12 cytokine family

Jones

Chaturvedi

Uyttenhove

et al. 2012

Molecular Immunology

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The heterodimeric IL-12 cytokine family is characterized by the sharing of three α (p19, p28, p35) and two β (p40 and Ebi3) subunits, and includes IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Ebi3) and IL-35 (p35/Ebi3). In this study, the dimerization interfaces of IL-12 family members were characterized, with emphasis on IL-35. Ebi3 and p35 subunits from human and mouse paired effectively with each other, indicating there is no species barrier to IL-35 dimerization and suggesting a conserved dimerization interface. Specific p35 residues that contribute to formation of the IL-12 interface were assessed for their contribution to the IL-35 interface, and candidate Ebi3 residues were screened for their contribution to both IL-27 and IL-35 interfaces. Several residues were identified as critical to the IL-12 or IL-27 interfaces. Conversely, no single mutation was identified that completely disrupts p35/Ebi3 pairing. Linear alanine scanning mutagenesis on both p35 and Ebi3 subunits was performed, focusing on residues that are conserved between the mouse and human proteins. Additionally, a structure-based alanine-scanning approach in which mutations were clustered based on proximitiy was performed on the p35 subunit. Both approaches suggest that IL-35 has distinct criteria for subunit pairing and is remarkabley less sensitive to structural perturbation than IL-12 and IL-27. Additionally, studies using a panel of anti-p35 and anti-Ebi3 antibodies indicate differential availability of epitopes within IL-12 family members that share these subunits, suggesting that IL-35 has distinct structural features, relative to IL-12 and IL-27. These results may be useful in future directed therapeutic targeting of IL-12 family members.

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“…The human and murine EpoRs have been cloned and consist of polypeptides with single membrane- spanning domains [18, 19, 20]. The cytokine receptor superfamily is characterized by the conservation of cysteines and a tryptophan-serine-x-tryptophan-serine (WSXWS) motif in the extracellular domain, as well as limited similarities in the cytoplasmic domain including box 1 and box 2 in the membrane proximal region [17, 18, 19, 20, 21]. No kinase or other enzyme motif has been identified in the cytoplasmic domains of members of the cytokine receptor superfamily.…”

Section: Erythropoietin Receptormentioning

confidence: 99%

Molecular Mechanisms of Erythropoietin Signaling

Cheung

Miller

2001

Nephron

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Erythropoietin is an obligatory growth factor for red blood cell production. The receptor for erythropoietin contains a single membrane-spanning domain with no intrinsic tyrosine kinase motifs. On binding to erythropoietin, the receptor dimerizes and activates multiple intracellular signaling molecules, including but not limited to JAK2, STAT5, PI 3-kinase, IRS-2, RAS, and Ca²⁺ channels. This review focuses on cytoplasmic signaling cascades involved in erythropoietin action.

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Saturation Mutagenesis of the WSXWS Motif of the Erythropoietin Receptor

Cited by 94 publications

References 60 publications

Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin

Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin

Distinct subunit pairing criteria within the heterodimeric IL-12 cytokine family

Molecular Mechanisms of Erythropoietin Signaling

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