Intramolecular Disulfide Bridges of the Prolactin Receptor (PRLR) Short Form are Required for its Inhibitory Action on the Function of the Long form of the Receptor

Abstract

The short form (SF), S1b, of the PRLR can silence PRL‐induced activation of gene transcription by the long form (LF). This results from LF/SF heterodimerization and the absence of cytoplasmic (C) sequences in the SF partner, which are essential for STAT activation. Dimerization of PRLR variants is ligand‐independent and PRL is a conformational modifier inducing JAK/STAT signaling. Mutagenesis of SF's four conserved Cys (S1bX), which form two intramolecular S‐S bonds within the extracellular (EC) subdomain‐1 (D1) PRL binding region, eliminated its inhibitory action on LF and its PRL‐induced JAK phosphorylation (JAK‐P). Since constitutive JAK‐P was observed in WT S1b, this change suggests that S‐S provides the conformation for JAK binding to box‐1 (adjacent to the trans‐membrane domain) in the C domain. BRET50 showed decreased heterodimeric association of LF/S1bX and increased affinity in homodimerization of S1bX, which favors LF dimerization and PRL‐induced signaling. Computer simulations of the EC based on the X‐ray crystal structure of PRLR showed less bending of D1 in S1bX. A positive grove that could be a motif of ligand recognition was occluded in S1bX. Additional inter‐monomer hydrogen bonds observed in D1 of S1bX may cause groove occlusion and higher homodimerization affinity of S1bx. The conformation of the PRLR provided by S‐S bridges is essential for the inhibitory action of SF.