title: Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization.
abstract: Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs). Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2). A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors. We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites. The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured. Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two. It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in. This interface is characterized by a binding "hot-spot" consisting of a core of three residues in ECD1 and two in ECD2. There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH. This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies.
authors.affiliation: Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, Cummings Life Sciences Center, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Bernat
authors.forename: Gabor
authors.initials: G
authors.lastname: Pal
authors.forename: Miao
authors.initials: M
authors.lastname: Sun
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006728:Hormones / Q000378:metabolism; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011986:Receptors, Somatotropin / Q000737:chemistry; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D006728:Hormones; D011986:Receptors, Somatotropin; D019382:Human Growth Hormone; D000409:Alanine