Article

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

Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, Eunice Kennedy Shriver National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2009; 29(10):2546-55. DOI: 10.1128/MCB.01716-08
Source: PubMed

ABSTRACT

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 (S-S) bonds within the extracellular subdomain 1 (D1) of S1b to its inhibitory function on the 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. BRET(50) (BRET(50) represents the relative affinity as acceptor/donor ratio required to reach half-maximal BRET [bioluminescence resonance energy transfer] values) 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 showed that minor changes in the tertiary structure of D1 upon S-S bond disruption propagated 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.

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    • ". , 2001 ) . Together with the growth hormone receptor ( GHR ) , the PRLR represents an archetype of class I cytokine receptors . Although these receptors were initially re - ferred to as ' homodimeric ' receptors ( O ' Sullivan et al . , 2007 ) , subsequent studies showed that various isoforms could actually heterodimerize ( Ross et al . , 1997 ; Xie et al . , 2009 ) and even antag - onize ( Trott et al . , 2003 ) . These receptors exhibit minimal structural complexity compared to other family members ( Bazan , 1990 ) . Their ECDs contain a single cytokine receptor homology ( CRH ) module consisting of two fibronectin type III domains called D1 and D2 ( Fig . 1A ) . In addition to residue I146 , t"
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