The high- and low-affinity receptor binding sites of growth hormone are allosterically coupled

Abstract

Growth hormone regulates its biological properties via a sequential hormone-induced receptor homodimerization mechanism. Using a mutagenesis-scanning analysis of 81 single and 32 pairwise double mutations, we show that the hormone's two spatially distal receptor binding sites (Site1 and Site2) are allosterically coupled. These allosteric effects are focused among a relatively few residues centered around the interaction between Asp-116 of the hormone and Trp-169 of the receptor in Site2. A rearrangement of this interaction triggered by mutations in Site1 produces both a major conformation and energetic reorganization of Site2, surprisingly without a reduction in overall binding affinity. Additionally, the data suggest a change in the conformational dynamics of several groups in Site2 that appear to be important in defining the Site2 interaction. Changes in binding energy of the affected Site2 residues usually range in magnitude from 3-to 60-fold, but in one case are as large as 10 4 .W ithin the cytokine super family, the growth hormone (GH)͞prolactin family of hormones and receptors is arguably the most extensively characterized system focused on establishing the structure-function basis for protein-protein interactions (1-5). These studies and those of related cytokine systems have been instrumental in defining structural similarities within the family that are deterministic for hormone action and regulation (6-12). The structure-based mechanisms by which these systems activate are similar (3,5,9,13). However, although these mechanisms are conceptually simple (hormone-induced receptor aggregation), it is becoming more appreciated that the molecular strategies that are used by each particular system are complex and hardly predictable (4,6,7,14).A hallmark of the GH-induced biological activity is a receptor aggregation process involving a receptor homodimerization through a mechanism that requires the two receptor extracellular domains (ECDs) to bind to the hormone in a highly regulated sequential order (13). The sequential order of binding is a consequence of the difference in binding affinities of the hormone's two spatially separated receptor binding sites. A highaffinity site, Site1, is always occupied first by the receptor ECD (R1) to form a stable 1:1 hormone-receptor intermediate complex [human GH (hGH)͞R1] (15). This 1:1 complex provides the structural platform for binding the second receptor ECD (R2) through two spatially distinct subsites, which are independently weak but together produce a tight R2 association. One subsite is located on the opposite face of the hormone to Site1 and is referred to as Site2 (Fig. 1). The second subsite, the so-called stem-stem contact, is formed by an extensive set of interactions between the C-terminal portions of the ECDs of the two receptor ECDs. Although it is clear that the two receptor ECDs are structurally integrated through this stem-stem contact, there is no direct evidence to suggest that the Site1 and Site2 binding sites on the hormone have any structural lin...

Obligate Ordered Binding of Human Lactogenic Cytokines

“…This interpretation extends and strengthens the data we have previously published for hPRL (9). Additionally, Walsh et al (10) have recently described a similar allosteric coupling of the two human growth hormone receptor binding sites in hGH, suggesting that a similar mechanism may govern hGH receptor binding. We have recently identified contiguous residues in hGH (15)-and hPRL (34)-constituting motifs that, when mutated, uncouple sites 1 and 2.…”

“…Previous surface plasmon resonance and Förster resonance energy transfer (FRET) data from our laboratory indicated that receptor binding induced a conformation change in the hormone associated with a functional coupling of sites1 and 2 (9). The observation that hPRL undergoes a conformational change upon receptor binding is consistent with observations of hGH or hPL binding the extracellular domain of the hPRL receptor (10,11) or when hGH binds the extracellular domain of the hGH receptor (12,13). The idea that the receptor binding events are functionally coupled also is consistent with the positive 1.7 Hill coefficient described by Hooper et al (3) in a heterologous ovine PRL/rat PRL receptor system.…”

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

“…However, no detailed molecular mechanism was formulated, and the extent to which PRL actually undergoes significant structural rearrangements when interacting with the receptor chain via BS1 has been a matter of some debate. In the GH system, it has been demonstrated that BS1 and BS2 are allosterically coupled in the sense that mutations in BS1 can induce conformational changes in BS2 (35). The recent publication of the crystal structure of the PRLR antagonist, PRLv3 (16), in combination with the present determination of the receptor-complexed structure of the closely related variant, PRLv2, provides a unique opportunity to scrutinize the structural rearrangements associated with receptor binding at BS1.…”

Long-range cooperative binding effects in a T cell receptor variable domain

“…Receptor homodimerization by the human growth hormone (hGH) has been shown to be allosterically regulated (37) and may represent an intermediate on such a continuum. The binding of the two receptor molecules to hGH occurs in a regulated and sequential manner.…”