MCB Accepts, published online ahead of print on 9 March 2009

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Mol. Cell. Biol. doi:10.1128/MCB.01716-08
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Intramolecular Disulfide Bonds of the Prolactin Receptor Short Form are Required for its Inhibitory Action on the Function of the Long form of the Receptor

Y-L. Xie, S. A. Hassan, A. M. Qazi, C. H. Tsai-Morris, and M. L. Dufau^*

Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institutes of Child Health and Human Development, Center for Molecular Modeling, DCB/CIT, National Institutes of Health, U.S. DHHS, Bethesda, Maryland 20892-4510

^* To whom correspondence should be addressed. Email: dufaum{at}mail.nih.gov.

The short form (S1b) of the prolactin receptor (PRLR) silences prolactin-induced activation of gene transcription by the PRLR long form (LF). The functional and structural contributions of two intramolecular disulfide-bonds (S-S) within the extracellular subdomain-1 (D1) of S1b to its inhibitory function on LF were investigated. Mutagenesis of the paired cysteines eliminated the inhibitory action of S1b. The expression of the mutated S1b (S1bX) on the cell surface was not affected, indicating native-like folding of the receptor. The constitutive JAK2 phosphorylation observed in S1b was not present in cells expressing S1bX, and JAK2 association was disrupted. BRET₅₀ showed decreased LF/S1bX heterodimeric-association and increased affinity in S1bX homodimerization, thus favoring LF homodimerization and prolactin-induced signaling. Computer modeling based on the PRLR crystal structure shows that minor changes in the tertiary structure of D1 upon S-S disruption propagate to the quaternary structure of the homodimer, affecting the dimerization interface. These changes explain the higher homodimerization affinity of S1bX, and provide a structural basis for its lack of inhibitory function. The PRLR conformation as stabilized by S-S bonds is required for the inhibitory action of S1b on prolactin-induced LF-mediated function and JAK2 association.