ABSTRACT
Crosstalk between opioid and adrenergic receptors is well characterized and due to interactions between second messenger systems, formation of receptor heterodimers, and extracellular allosteric binding regions. Both classes of receptors bind both sets of ligands. We propose here that receptor crosstalk may be mirrored in ligand complementarity. We demonstrate that opioids bind to adrenergic compounds with micromolar affinities. Additionally, adrenergic compounds bind with micromolar affinities to extracellular loops of opioid receptors while opioids bind to extracellular loops of adrenergic receptors. Thus, each compound type can bind to the complementary receptor, enhancing the activity of the other compound type through an allosteric mechanism. Screening for ligand complementarity may permit the identification of other mutually-enhancing sets of compounds as well as the design of novel combination drugs or tethered compounds with improved duration and specificity of action.
Subject(s)
Adrenergic Agonists/chemistry , Analgesics, Opioid/chemistry , Drug Development , Receptors, Adrenergic/chemistry , Receptors, Opioid/chemistry , Adrenergic Agonists/pharmacology , Analgesics, Opioid/pharmacology , Drug Development/methods , Humans , Kinetics , Ligands , Models, Biological , Peptides/chemistry , Peptides/metabolism , Protein Binding , Receptors, Opioid/agonists , Structure-Activity RelationshipABSTRACT
Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.
Subject(s)
Adrenergic Agonists/metabolism , Enkephalin, Methionine/metabolism , Morphine/metabolism , Protein Interaction Domains and Motifs , Receptors, Opioid, mu/metabolism , Acetylcholine/chemistry , Acetylcholine/metabolism , Adrenergic Agonists/chemistry , Amino Acid Sequence , Animals , Enkephalin, Methionine/chemistry , Histamine/chemistry , Histamine/metabolism , Humans , Methionine/chemistry , Methionine/metabolism , Mice , Morphine/chemistry , Protein Binding , Receptors, Adrenergic, alpha-1/chemistry , Receptors, Adrenergic, alpha-1/metabolism , Receptors, Opioid, mu/chemistry , Spectrophotometry, UltravioletABSTRACT
Par-4 is a unique proapoptotic protein with the ability to induce apoptosis selectively in cancer cells. The X-ray crystal structure of the C-terminal domain of Par-4 (Par-4CC), which regulates its apoptotic function, was obtained by MAD phasing. Par-4 homodimerizes by forming a parallel coiled-coil structure. The N-terminal half of Par-4CC contains the homodimerization subdomain. This structure includes a nuclear export signal (Par-4NES) sequence, which is masked upon dimerization indicating a potential mechanism for nuclear localization. The heteromeric-interaction models specifically showed that charge interaction is an important factor in the stability of heteromers of the C-terminal leucine zipper subdomain of Par-4 (Par-4LZ). These heteromer models also displayed NES masking capacity and therefore the ability to influence intracellular localization.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Amino Acid Sequence , Apoptosis/physiology , Apoptosis Regulatory Proteins/genetics , Cell Nucleus/metabolism , Humans , Protein Conformation , Protein MultimerizationABSTRACT
It has long been known in spectroscopy that light not passing through a sample, but reaching the detector (i.e., stray light), results in a distortion of the spectrum known as absorption flattening. In spectroscopy with crystals, one must either include such stray light or take steps to exclude it. In the former case, the derived spectra are not accurate. In the latter case, a significant amount of the crystal must be masked off and excluded. In this paper, we describe a method that allows use of the entire crystal by correcting the distorted spectrum.
Subject(s)
Crystallography/methods , Purple Membrane/chemistry , Scattering, Radiation , Spectrum Analysis/methods , Kinetics , LightABSTRACT
Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteins via its C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7â Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to be P41212. The unit-cell parameters are a = b = 115.351, c = 123.663â Å, α = ß = γ = 90°.
