Functional Epitopes for Site 1 of Human Prolactin

Abstract

Human prolactin (hPRL) binds two human prolactin receptor molecules, creating active heterotrimeric complexes. Receptors bind dissimilar hormone surfaces termed site 1 and site 2 in an obligate ordered process. We sought to map the functional epitopes in site 1 of hPRL. Extensive alanine mutagenesis (102 of the 199 residues) showed approximately 40% of these mutant hPRLs changed the ΔG for site 1 receptor binding. Six of these residues are within 3.5 Å of the receptor and form the site 1 functional epitopes. We identified a set of noncovalent interactions between these six residues and the receptor. We identified a second group of site 1 residues that are between 3.5 and 5 Å from the receptor where alanine mutations reduced the affinity. This second group has noncovalent interactions with other hormone residues and stabilized the topology of the functional epitopes by linking these to the body of the protein. Finally, we identified a third group of residues that are outside site 1 (>5 Å) and extend to site 2 and whose mutation to alanine significantly weakened receptor binding at site 1 of prolactin. These three groups of residues form a contiguous structural motif between sites 1 and 2 of human prolactin and may constitute structural features that functionally couple sites 1 and 2. This work identifies the residues that form the functional epitopes for site 1 of human prolactin and also identifies a set of residues that support the concept that sites 1 and 2 are functionally coupled by an allosteric mechanism.

Mutant Prolactin Receptor and Familial Hyperprolactinemia

“…The familial idiopathic hyperprolactinemia–associated His188Arg substitution in the prolactin receptor, which leads to prolactin insensitivity, is located in the extracellular domain and affects a histidine residue, which is within the high-affinity ligand-binding interface and is critical for prolactin-receptor function. 13,18 This PRLR mutation did not result in altered immune function, a finding that is consistent with findings in Prlr -null mice that indicate that prolactin is not an obligate lymphopoietic hormone. 21,22 …”

“…13,18,19 Prolactin interacts with each extracellular domain of the dimeric prolactin receptor, first by means of a high-affinity (nanomolar range) binding site 1, and second, by means of a low-affinity (micromolar range) binding site 2 (Fig. 1D).…”

Mutant Prolactin Receptor and Familial Hyperprolactinemia

“…The familial idiopathic hyperprolactinemia-associated His188Arg substitution in the prolactin receptor, which leads to prolactin insensitivity, is located in the extracellular domain and affects a histidine residue, which is within the highaffinity ligand-binding interface and is critical for prolactin-receptor function. 13,18 This PRLR mutation did not result in altered immune function, a finding that is consistent with findings in Prlr-null mice that indicate that prolactin is not an obligate lymphopoietic hormone. 21,22 To date, one other nonsynonymous PRLR variant, causing a change from isoleucine to leucine at codon 146 in the extracellular domain of the mature protein and resulting in an increased basal JAK2-STAT5 signaling in vitro, has been reported in women with benign Europe PMC Funders Author Manuscripts breast fibroadenomas.…”

“…13,18,19 Prolactin interacts with each extracellular domain of the dimeric prolactin receptor, first by means of a high-affinity (nanomolar range) binding site 1, and second, by means of a lowaffinity (micromolar range) binding site 2 ( Fig. 1D).…”

“…1D). 11,14,18 His188 is found at the interface of the high-affinity binding site 1, 12,13,18 where it forms a polar contact (i.e., a noncovalent bond) with prolactin ( Fig. 1E) and contributes to a four-member histidine ring, formed by His188 in the prolactin receptor and three histidines of prolactin (His27, His30, and His180), which stabilizes ligand binding and determines the pH dependence of receptor activation.…”