Receptor signaling in the growth hormone (GH)-growth hormone receptor (GHR) system is controlled through a sequential two-step hormone-induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of the second ECD (ECD2) to the stable preassociated 1 : 1 GH/ECD1 complex on the cell surface. To determine the energetics that governs this step, the binding kinetics of 38 single-and double-alanine mutants in the hGH Site2 contact with ECD2 were measured by using trimolecular surface plasmon resonance (TM-SPR). We find that the Site2 interface of hGH does not have a distinct binding hot-spot region, and the most important residues are not spatially clustered, but rather are distributed over the whole binding surface. In addition, it was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues. Residues that show little effect or even improved binding on substitution with alanine, when paired with D116A-hGH, display significant negative cooperativity. Because most of these pairwise mutated residues are spatially separated by Ն10 Å, this indicates that the Site2 binding interface of the hGH-hGHR ternary complex displays both structural and energetic malleability.

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...

Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by 1) adding long branched hydrophilic linkers between the substrate and the surface, 2) changing the orientation of peptides relative to the surface, and 3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates.

Turtle visual cortex has three layers and receives direct input from the dorsolateral geniculate complex of the thalamus. The outer layer 1 contains several populations of interneurons, but their physiological properties have not been characterized. This study used intracellular recording methods followed by filling with Neurobiotin to characterize the morphology and physiology of two populations of layer 1 interneurons. Subpial cells have somata positioned in the outer third of layer 1 and dendrites confined within the band of geniculate afferents that runs from lateral to medial across visual cortex. Their dendrites are composed of a sequence of many beads or varicosities separated by intervaricose segments. They have membrane time constants of tau(o) = 45.5 +/- 5.2 ms and electrotonic lengths of 1.1 +/- 0.2. Subpial cells show spike rate adaptation in response to intracellular current pulses. Stellate cells have somata located in the inner two-thirds of layer 1 and, less frequently, in layers 2 and 3. Their dendrites extend in a stellate configuration across the cortex. They are smooth or sparsely spiny, but never bear distinct varicosities. They have membrane time constants of tau(o) = 155.1 +/- 12 ms and electrotonic lengths of 3.8 +/- 0.5. They show little spike rate adaptation in response to intracellular current pulses. The positions of the two populations of cells in visual cortex and their physiological properties suggest that subpial cells may participate in a feedforward inhibitory pathway to pyramidal cells, whereas stellate cells are involved in feedback inhibition to pyramidal cells.

A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immuno-chemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via non-covalent biotin-streptavidin interactions. This non-covalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immuno-chemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC50 (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immuno-chemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable and inexpensive route to the discovery of small molecule drug leads.