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.
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.
Xie
Y-L
YL
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.
Hassan
S A
SA
Qazi
A M
AM
Tsai-Morris
C H
CH
Dufau
M L
ML
D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011388:Prolactin / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011981:Receptors, Prolactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004220:Disulfides; D020033:Protein Isoforms; D011981:Receptors, Prolactin; D011388:Prolactin; D053614:Janus Kinase 2; D003545:Cysteine