Identification of ligand binding determinants of the prolactin receptor.
The prolactin (PRL) receptor, a lactogen- and primate somatogen-binding protein, is a member of an expanding superfamily (cytokine/growth hormone (GH)/PRL) of single membrane-spanning receptors. Two features commonly shared among this group of proteins are the presence of two pairs of cysteines, generally found in the N-terminal region of the extracellular domain, and a WSxWS (WS) motif, frequently located proximal to the transmembrane domain. We have recently shown the 4 cysteines to be critical to the maintenance of the structural and functional integrity of the PRL-receptor. In the present study, we prepared a set of eight chimeric rat PRL/human GH receptors and several alanine mutants, to assess the importance of the Cys-rich domain (residues 12-68) in confering specificity to PRL binding. The role of the WS motif in high affinity binding was also investigated. Binding of 125I-labeled ovine PRL or human GH to membrane preparations from COS-7 cells transiently expressing the mutant receptors have defined a region within the first disulfide loop (residues Arg13, Asp16, Glu18) and the set of lactogen-specific sequences between the two pairs of cysteines as key determinants of PRL-binding specificity, which converge to form a patch on a two-dimensional model of the PRL receptor. We also demonstrate that, although PRL- and GH-specific determinants overlap in certain areas, they are not identical. Finally, substitution of the WS motif with alanine residues precludes high affinity binding to ovine PRL and human GH and suggests that this structural element may provide a target site for the interaction of an accessory protein necessary for the formation of a high-affinity receptor complex.
Rozakis-Adcock
M
M
Laboratory of Molecular Endocrinology, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Kelly
P A
PA
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011981:Receptors, Prolactin / Q000235:genetics / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012756:Sheep; D013379:Substrate Specificity
D008024:Ligands; D011981:Receptors, Prolactin; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D000409:Alanine