Receptor-ligand binding assays: technologies and applications

Receptor-ligand interactions play a crucial role in biological systems and their measurement forms an important part of modern pharmaceutical development. Numerous assay formats are available that can be used to screen and quantify receptor ligands. In this review, we give an overview over both radioactive and non-radioactive assay technologies with emphasis on the latter. While radioreceptor assays are fast, easy to use and reproducible, their major disadvantage is that they are hazardous to human health, produce radioactive waste, require special laboratory conditions and are thus rather expensive on a large scale. This has led to the development of non-radioactive assays based on optical methods like fluorescence polarization, fluorescence resonance energy transfer or surface plasmon resonance. In light of their application in high-throughput screening environments, there has been an emphasis on so called "mix-and-measure" assays that do not require separation of bound from free ligand. The advent of recombinant production of receptors has contributed to the increased availability of specific assays and some aspects of the expression of recombinant receptors will be reviewed. Applications of receptor-ligand binding assays described in this review will relate to screening and the quantification of pharmaceuticals in biological matrices.     

Receptor-ligand binding in the cell-substrate contact zone: a quantitative analysis using CX3CR1 and CXCR1 chemokine receptors

Receptor-ligand binding analyses have generally used soluble components to measure thermodynamic binding constants. In their biological context, adhesion receptors bind to an immobile ligand and the binding reaction is confined to the cell-substrate contact zone. We have developed a new procedure based on the spinning disk technology to measure the number of receptor-ligand bonds in the contact zone. Application of this methodology to the CX3CR1-fractalkine and the CXCR1-IL-8 receptor-ligand systems demonstrated that the level of binding to an immobilized ligand is reduced by several orders of magnitude in comparison to solution binding. A comparison of the solution binding and contact zone binding constants shows that the effect of ligand immobilization was similar for each system. In contrast, although the CXCR1-IL-8 bond had the higher affinity, the average bond strength was only 10% of that for the CX3CR1 bond. Because fractalkine can be expressed as a cell surface-bound protein, CX3CR1 has been proposed to function as an adhesion receptor. The higher bond strength suggests that the bond architecture has also evolved to serve an adhesion function.     

Indicted: worms caught using steroids

Three recent papers provide new insights into endocrinology in the worm Caenorhabditis elegans. These studies identify natural steroid ligands for the DAF-12 nuclear receptor, define a new enzyme in the hormone biosynthetic pathway, and clarify the role of endocrine signaling in adult longevity.     

Proteins: caught in the trap

Two independent groups have recently devised innovative methods using light to trap and manipulate particles as small as proteins.     

Membrane fusion: caught in a trap

SNAREs are small coiled-coil proteins required for specific membrane fusion events in eukaryotic cells. Recent evidence points to the existence of an inhibitory class of SNAREs, i-SNAREs, which prevent incorrect fusions from occurring, adding a further layer of regulation to the process of membrane docking and fusion.     

DNA manipulators: caught in the act

DNA is a dynamic molecule that undergoes constant changes in the cell through interactions with numerous proteins. Several classes of enzyme are specialized in promoting DNA rearrangements, including site-specific recombinases, DNA helicases, transposases and DNA topoisomerases. Recent structures of protein-DNA reaction intermediates trapped in various states of DNA remodeling, complemented by biochemical and biophysical functional studies, have enhanced our understanding of their respective mechanistic pathways.     

Actin finally matures: uncovering machinery and impact

Actin, one of the most abundant proteins in nature and a key component of the cytoskeleton, undergoes a unique multistep N-terminal (Nt) maturation. In a recent report, Haahr et al. identified actin maturation protease (ACTMAP) as the dedicated actin aminopeptidase and showed that its absence is associated with abnormal muscle physiology.     

MTT overview: Monolithic finally found a congregation

After all these years of monolithic people “preaching only to the choir”, MTT-S (the annual IEEE International Microwave Symposium, May 7–11, Dallas) marked a turning point for many microwave and radio science engineers.     

Ethylene signaling: the MAPK module has finally landed

Ethylene hormone responses are negatively regulated by the CTR1 protein, which has similarity to mitogen-activated protein kinase kinase kinases (MAPKKKs). Because of this similarity, it has long been speculated that ethylene signal transduction involves a MAPK cascade. Now, a recent paper provides compelling evidence for an ethylene-activated MAPK pathway. The implication is that CTR1 and the newly identified MAPKK and MAPKs comprise a MAPK module that regulates ethylene responses in plants.     

Receptor-ligand interactions and tolerance induction in mature virgin B cells

The membrane events and the nature of the receptors involved in the induction of thymus-independent high-zone tolerance were investigated. Tolerance was induced in vitro by incubating cells for the 4 hr with DNP-polysaccharide conjugate. The degree of tolerance was estimated by determining the subsequent cellular response to antigenic challenge in vitro. Treatment with agents that inhibited energy metabolism, membrane fluidity, and movement of membrane receptors all inhibited the induction of tolerance. Agents which affect the integrity of cytoskeletal elements also interfered with tolerance induction. Taken together these results indicate that the induction of high-zone thymus-independent tolerance is an active process involving at least some aspects of antigen induced receptor modulation. The specific receptor involved appears to be IgM since tolerance could be induced by exposing cells first to subtolerogenic doses of antigen and then to antibodies specific for the IgM receptor.     

Gene therapy: light is finally in the tunnel

After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber’s congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adeno-associated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.     

Secretion: kiss and run caught on film

Recent results have provided graphic support for the hypothesis that vesicle secretion involves a 'kiss-and-run' mechanism. Evanescent field microscopy has shown that, during exocytosis, intravesicular markers escape without collapse of the vesicular membrane into the surface membrane and that the empty vesicle is immediately retrieved back into the cell.     

Actin nucleation: cortactin caught in the act

A variety of activators stimulate Arp2/3 complex to nucleate branched actin filament structures. New results provide important biochemical and structural information for activation by the proteins cortactin and N-WASP.     

Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195. Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.     

Organelle division: Self-assembling GTPase caught in the middle

Recent findings indicate that the dynamin GTPase helps to divide animal and fungal mitochondria, and that the tubulin-like FtsZ GTPase is involved in division of, not only most bacteria, but also chloroplasts and probably mitochondria of unicellular eukaryotes.     

The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness

Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross‐reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4‐dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross‐reactants are themselves bound specifically—close derivatives of these cross‐reactants show very low or no binding to SPE7. It has been suggested that cross‐reactivity is simply due to “hydrophobic stickiness”, nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross‐reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1–8. By comparing the sequences and binding patterns of SPE7 and B1–8, we address the relationship between affinity maturation, specificity, and cross‐reactivity.     

DNA topoisomerases: single gyrase caught in the act

A recent study has analysed the action of bacterial DNA gyrase on a single substrate DNA molecule, discriminating the initial DNA wrapping and subsequent supercoiling steps in the reaction cycle.     

Primary piRNA biogenesis: caught up in a Maelstrom

Precursors for most Piwi‐interacting RNAs (piRNAs) are indistinguishable from other RNA polymerase II‐transcribed long non‐coding RNAs. So, it is currently unclear how they are recognized as substrates by the piRNA processing machinery that resides in cytoplasmic granules called nuage. In this issue, Castaneda et al (2014) reveal a role for the nuage component and nucleo‐cytoplasmic shuttling protein Maelstrom in mouse piRNA biogenesis.