Formyl peptide chemotaxis receptors on the rat neutrophil: Experimental evidence for negative cooperativity

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[ 3 H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analyses of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P < 0.1). In addition, Hill coefficients of 0.80 ± 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[ 3 H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[ 3 H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone, In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[ 3 H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[ 3 H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38446/1/240270406_ftp.pd

Interaction between [3H]ethylketocyclazocine and [3H]etorphine and opioid receptors in membranes from rat brain a kinetic analysis

Scatchard analysis of the binding to opioid receptors of pHlethylketocyclazocine ([3H]EKC) and [3H]etorphine at equilibrium yielded biphasic plots and computer fitting of the data resulted in a minimal model of two independent saturable binding sites. The KD values for the high- and low-affinity sites were 0.58 and 38 nM for [3H]EKC, and 0.13 and 22 nM for [3H]etorphine. The corresponding density of binding sites was 157 and 418 fmol/mg protein for [3H]EKC, and 220 and 289 fmol/mg protein for [3H]etorphine. The KD values calculated from the association and dissociation rate constants corresponded to those observed at equilibrium. In the course of equilibrium binding, various opioids competed with [3H]ekc and [3H]etorphine preferentially at the high-affinity opioid receptor sites. No difference between the competition patterns of putative [mu] and [kappa] ligands was observed.The kinetics of association and dissociation of [3H]EKC and [3H]etorphine revealed that the apparently homogeneous high-affinity binding site observed at equilibrium consisted of two components characterized by their fast and slow equilibrium times, respectively. While none of the [mu] and [kappa] opiates investigated altered the rate of dissociation of [3H]EKC or [3H]etorphine, in the presence of sodium ions the rapidly dissociating binding component of [3H]etorphine became refractory to inhibition by [mu] but not [kappa] agonists. The results underline the advantages of evaluating both equilbrium binding and the kinetics of ligand-receptor interactions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26219/1/0000299.pd

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design.Peer reviewe