title: Mechanism for ordered receptor binding by human prolactin.
abstract: Prolactin, a lactogenic hormone, binds to two prolactin receptors sequentially, the first receptor binding at site 1 of the hormone followed by the second receptor binding at site 2. We have investigated the mechanism by which human prolactin (hPRL) binds the extracellular domain of the human prolactin receptor (hPRLbp) using surface plasmon resonance (SPR) technology. We have covalently coupled hPRL to the SPR chip surface via coupling chemistries that reside in and block either site 1 or site 2. Equilibrium binding experiments using saturating hPRLbp concentrations show that site 2 receptor binding is dependent on site 1 receptor occupancy. In contrast, site 1 binding is independent of site 2 occupancy. Thus, sites 1 and 2 are functionally coupled, site 1 binding inducing the functional organization of site 2. Site 2 of hPRL does not have a measurable binding affinity prior to hPRLbp binding at site 1. After site 1 receptor binding, site 2 affinity is increased to values approaching that of site 1. Corruption of either site 1 or site 2 by mutagenesis is consistent with a functional coupling of sites 1 and 2. Fluorescence resonance energy transfer (FRET) experiments indicate that receptor binding at site 1 induces a conformation change in the hormone. These data support an "induced-fit" model for prolactin receptor binding where binding of the first receptor to hPRL induces a conformation change in the hormone creating the second receptor-binding site.
authors.affiliation: The Ohio State Biochemistry Program and Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Umasundari
authors.initials: U
authors.lastname: Sivaprasad
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Canfield
authors.forename: Charles L
authors.initials: CL
authors.lastname: Brooks
mesh_terms: D001667:Binding, Competitive / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D003911:Dextrans / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011981:Receptors, Prolactin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D003911:Dextrans; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D011388:Prolactin; D008715:Methionine; D003545:Cysteine; D005998:Glycine