Role of the polymorphic residues in HLA-DR molecules in allele-specific binding of peptide ligands.

Analysis of peptide binding to a set of HLA-DR alleles has allowed the proteins to be segregated into functional subsets, depending on the amino acids at positions 57 and 86 of the beta-chain. DR proteins with glycine at 86 beta and aspartic acid at 57 beta bound a simplified peptide with significantly lower IC50 values than alleles that did not have this combination of amino acids. The size of the amino acid at 86 beta seemed to modify the steric requirements for the single most important side chain of the peptide. Within each of the four subgroups, other polymorphic amino acids define allele-specific binding requirements. These were explored by analyzing the ability of eight different DR alleles to bind 13 known T cell determinants. The side chains in the peptides that seemed to be responsible for allele specificity were determined by correlating their common structural features with complementary polymorphic residues in the binding site. The importance of these residues was tested by incorporating them into a polyalanine backbone, and was confirmed by the ability of these residues to transfer allele specificity to these simplified analogues. Even though polymorphic contacts affected peptide affinity, the majority of the free energy of binding in all cases arose from interactions with the peptide backbone and the single hydrophobic amino acid at the third position. These constraints seem to orient all peptides in a similar location, forcing them to adopt a closely related conformation in the binding site. The corresponding side chain in each peptide contacts the same pocket in the binding site, regardless of the allele. This apparent similarity should allow any DR allele to be analyzed by extrapolation from the DR1 crystal structure.

Temperature effects on alpha 2-adrenergic receptor-Gi interactions.

The effect of temperature on the binding of alpha 2-adrenergic agonists and antagonists to human platelet membranes was studied. Equilibrium binding of the alpha 2 antagonist, [3H]yohimbine, was affected minimally, whereas the rate of dissociation changed 40-fold over a temperature range of 5-35 degrees. The antagonist dissociation rates were characterized by a linear Arrhenius plot and an activation energy of 20.5 kcal/mol. The equilibrium binding of the full alpha 2 agonist, [3H]UK 14,304 [5-bromo-6-N-2-4,5-dihydroimidazolyl)quinoxaline tartrate] showed a 50% decrease in Bmax at 5 degrees as well as a 2-fold decrease in affinity. The kinetics of [3H]UK 14,304 binding were affected more significantly by decreases in temperature. The agonist exhibited fast and slow phases of binding. The fast binding was minimally sensitive to temperature in the range of 0-30 degrees with only a 6-fold change in rate. The slow binding rates changed nearly 100-fold over the same temperature range. Also, the slow rate of agonist binding was characterized by a nonlinear Arrhenius plot with a "break" at approximately 17 degrees, which was found previously to be the phase transition temperature of platelet membrane lipids [Lohse et al., Molec. Pharmac. 29, 228 (1986)]. Despite the reduction of high affinity [3H]UK 14,304 binding at 5 degrees, approximately half of the binding remained sensitive to guanine nucleotides. The data are interpreted in the context of a model in which the fast agonist binding represents a bimolecular interaction of ligand with two pre-existing conformations of the alpha 2 receptor, one coupled to Gi and the other permanently uncoupled. The slow binding of agonist appears to require protein diffusion in the lipid membrane or a protein conformational change which is dependent on the lipid environment.

Mechanism of agonist and antagonist binding to alpha 2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex.

The alpha 2 adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with Ni, a guanine nucleotide binding protein. The early steps involved in the activation of the alpha 2 AR by agonists and the subsequent interaction with Ni are poorly understood. In order to better characterize these processes, we have studied the kinetics of ligand binding to the alpha 2 AR in human platelet membranes on the second time scale. Binding of the alpha 2 antagonist [3H]yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 X 10(5) M-1 s-1 and a dissociation rate constant of 1.11 X 10(-3) s-1. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the alpha 2 adrenergic full agonist [3H]UK 14,304 was characterized by two binding affinities: Kd1 = 0.3-0.6 nM and Kd2 = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of [3H]UK 14,304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics on [3H]UK 14,304 concentration revealed a fast phase with an apparent bimolecular reaction constant kappa + of 5 X 10(6) M-1 s-1. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the "ternary complex" binding mechanism. In the model which best accounted for the data, (1) approximately one-third of the alpha 2 adrenergic receptor binds agonist with low affinity and is unable to couple with a guanine nucleotide binding protein (N protein), (2) approximately one-third is coupled to the N protein prior to agonist binding, and (3) the remainder interacts by a diffusional coupling of the alpha 2 AR with the N protein or a slow, ligand-independent conformational change of the alpha 2 AR-N protein complex. The rates of interaction of liganded and unliganded receptor with N protein are estimated.

Vascular interactions of serotonin and norepinephrine in renal hypertensive rabbits.

Vascular responses to norepinephrine (NE) are potentiated in the presence of serotonin (5-HT) and are attenuated by previous exposure to 5-HT. This study was designed to determine whether alterations in the interactions of these agonists occur in renal hypertension. Helical strips of femoral artery from two-kidney, one clip (2K1C) hypertensive and control rabbits were mounted in muscle baths for isometric force recording. Arterial strips from 2K1C rabbits were more sensitive to 5-HT than were those of control rabbits. The sensitivities of these two groups of vessels to NE were not significantly different. The 5-HT potentiation of NE was present at lower 5-HT concentrations in vessels from 2K1C rabbits than in those from control rabbits. Previous exposure to 5-HT attenuated NE responses of arteries from 2K1C less than it did those of control rabbits. Although 5-HT had little effect on loading of cellular stores of calcium in the resting muscle, previous 5-HT exposure attenuated calcium influx during an NE response, and this attenuation was not as great in vascular smooth muscle from the hypertensive animal as it was in that from the normotensive control. These results indicate that in vascular smooth muscle cells from 2K1C rabbits the potentiating action of 5-HT is increased, due to the increased sensitivity to this agonist, and the attenuating action is decreased due to a greater calcium influx during NE stimulation.

Agonist and antagonist binding to alpha 2-adrenergic receptors in purified membranes from human platelets. Implications of receptor-inhibitory nucleotide-binding protein stoichiometry.

The agonist- and antagonist-binding properties of the alpha 2-adrenergic receptor in a purified plasma membrane preparation from human platelets were determined both by direct binding of radiolabeled ligands and by competition with the labeled alpha 2-antagonist, [3H] yohimbine. Binding of [3H]yohimbine was characterized by a single high affinity binding site (Kd = 6.2 +/- 1.4 nM, Bmax = 507 +/- 53 fmol/mg). In direct binding studies, the imidazoline full alpha 2-agonist, [3H]-5-bromo-6-N(2-4,5-dihydroimidazolyl)quinoxaline ([3H] UK 14,304), bound to only one quantifiable high affinity site (Kd = 0.88 +/- 0.17 nM), representing 65 +/- 6% of the number of [3H]yohimbine sites. Binding of the partial agonist [3H]-p-aminoclonidine (PAC) showed nonlinear Scatchard plots. Analysis according to a model of multiple independent binding sites showed the data to be consistent with two sites (Kd1 = 0.62 +/- 0.18 nM and Kd2 = 7.9 +/- 1.4 nM). The high affinity site corresponded to 15 +/- 6% and the low affinity site corresponded to 39 +/- 6% of the number of [3H]yohimbine sites. Competition for binding of the alpha 2-antagonist, [3H]yohimbine, with nonradiolabeled ligands revealed a single affinity for yohimbine. In contrast, competition for [3H]yohimbine binding by the full agonist UK 14,304 and epinephrine is best fit by a model with two independent binding sites. The partial agonist PAC was best characterized by a model with three distinct binding sites. The full agonists UK 14,304 and epinephrine inhibited adenylate cyclase approximately 30%, whereas PAC produced only 12% inhibition. The inhibitory guanine nucleotide-binding protein (Ni) with Mr 40,700 was the sole pertussis toxin substrate in the purified membranes. It was quantitated by pertussis toxin-catalyzed [32P]ADP ribosylation in cholate extracts. There is a 20- to 100-fold excess of Ni over alpha 2-adrenergic receptors. Comparisons made between the experimental data for agonist binding and theoretical predictions of the simple ternary complex model suggest that there is compartmentalization of Ni and/or that the alpha 2 receptors are heterogeneous.

Relaxation of rat tail artery to electrical stimulation.

Rat tail artery strips relax in response to electrical stimulation (0.1-8Hz, 9V, 1.0msec) following contraction induced by norepinephrine (5.9 X 10(-7)M). The relaxation is not altered by treatment of the strips with atropine, propranolol, tetrodotoxin or indomethacin nor by chemical denervation with 6-hydroxydopamine. Incubation of strips in calcium-free solution reduced the contractile response to norepinephrine and blocked relaxation in response to 4Hz electrical stimulation. Histamine antagonists (H2 receptor subclass: cimetidine, metiamide) inhibited the relaxation to electrical stimulation in a dose-dependent manner. These results suggest that relaxation to electrical stimulation in rat tail artery is modulated by calcium and by the H2 subclass of histamine receptors.

Ca2+, histamine antagonists and relaxation to electrical impulses in dog coronary artery.

Isolated dog coronary arteries relax in response to electrical stimulation (0.1-8.0 Hz, 9 V, 1.0 ms) following contraction induced by serotonin. Cimetidine, metiamide and ranitidine inhibited this relaxation. The relaxation was not blocked by pyrilamine. Reducing the concentration of Ca+ (0.1 mM) decreased the rate of relaxation whereas relaxation was more rapid when the Ca2+ concentration was increased (3.2 mM). These results suggest that relaxation to electrical stimulation is modulated by Ca2+ and by the H2-subclass of histamine receptors.