Receptor:ligand interactions account for numerous reactions critical to biochemistry and molecular biology. While students are typically exposed to some examples, such as hemoglobin binding of oxygen and signal transduction pathways, the topic could easily be expanded. Theory and kinetic analysis, types of receptors, and the experimental assay techniques should be included to properly prepare students for careers in research, medicine, or professional programs. In this article we offer a range of material for teaching these concepts to students.
WHY TEACH RECEPTOR ANALYSIS? A HOLISTIC APPROACH TO BIOCHEMISTRY
As biochemists, we teach our students about the many molecules involved in cellular processes: DNA, RNA, proteins, lipids, and carbohydrates. Tremendous detail is provided about metabolism and other cellular processes, and as part of that, students learn detailed theory about the regulation of enzymes and kinetic analysis, which is critical to understanding cellular processes and control. In a similar vein, signal transduction is introduced as it induces enzyme activation and the transport of small molecules such as calcium ions.
Vast amounts of research in industry and academia are geared toward receptor understanding and manipulation, so it is clear that this field needs greater emphasis at the undergraduate level. Proteins binding to DNA, co-activators interacting with enzymes, antibodies, and neurotransmitters binding to synaptic receptors all require some simple knowledge of molecular interaction at the chemical level. While some students learn receptor analysis in other courses, the material is similar to the coverage given to enzyme kinetics and could easily be included in a biochemistry course and textbooks to serve a greater portion of the undergraduate population.
In this article we present a framework for teaching biochemistry to undergraduate students. The authors teach receptor theory and application from two different standpoints to two different audiences: biochemistry and psychology classes that include both majors and non-majors. We have taught entire courses on receptor theory and have included limited lectures within more general classes. Examples of receptor types and activities, experimental theory on the characterization of receptors, and receptor regulation are included in this article.
QUANTITATIVE AND QUALITATIVE ANALYSIS OF RECEPTORS
It is straightforward to introduce receptor analysis to students. The quantitative and qualitative characterization of receptors is similar to enzyme kinetics. Because there are so many similarities between enzymes and receptors, students can readily learn the material after they have mastered the sections on enzyme kinetics and regulation. Learning about receptor analysis may even improve student understanding of enzymatic assays, results, and interpretation.
The similarities between enzymes and receptors can be easily observed by direct comparison as shown in Table I. Instead of enzyme:substrate (E:S)11 binding, a ligand binds to a receptor (R:L). (The ligand is frequently referred to as a hormone or drug in other disciplines.) Instead of a product, there is usually (although not always) a signal or event that can be measured as a result of the R:L interaction. The binding of a receptor to ligand is a reversible event as is the binding of substrate by enzyme. The formation of the complex is dependent on pH, temperature, ions, and other molecules. Like E:S complexes, the affinity of the binding can be measured as well as the level of saturation of the receptor by the ligand. Molecules that bind to a receptor but do not produce an effect are called antagonists, whereas those that bind to a receptor and induce a response are termed agonists.
Similar to enzymes, the molecules bound by receptors may be very small, such as a small sugar or amino acid, or may be very large, such as another protein. DNA binding a protein is a type of R:L complex as is the insulin receptor binding of insulin.
The largest problem encountered in studying receptors is that determining quantitative and qualitative data is technically difficult. If a specific event can be measured, then the response is used to determine the relevant data. The neuroreceptors are good examples of this phenomenon. But if there is not an event that can be observed, the formation of a R:L complex must be measured. While it is not as fleeting as a transition state, the act of recovering and measuring the complex can alter its presence and skew results. Designing an assay that can accurately determine the R:L complex in a quantitative manner under physiological conditions can be difficult, if not impossible. Various techniques have been developed. The specific binding must be “teased out” from binding that is not of high affinity and may not be “real.” More than one dismayed scientist has characterized the binding of a ligand to a test tube wall! Excellent sources of material for teaching the types of plots and results used in receptor research can be found in texts  as well as web sites .
A receptor will bind most ligands in both a specific and nonspecific manner. The total binding is the combination of the two types, and therefore specific binding (BS) is determined as a part of the total binding (BT). Nonspecific binding (BN)is considered to have infinite sites and be of low affinity, whereas specific binding is considered saturable and high affinity (see Fig. 1). For example, insulin can bind with high affinity to insulin receptors. However, in addition to this binding, insulin will also bind with an infinite number of “false” receptors but at lower affinity. The false receptors can include other proteins, improper sites on the receptor itself, binding to test tubes, or anomalous binding to the medium used to separate the complex from the free ligand. The total binding is a sum of the specific and nonspecific binding. The simplest way to measure the total binding is directly in the presence of receptor and ligand under varying conditions. Typically the ligand is labeled by radioactivity or another tag such as fluorescence. For example, a radioactive ligand (*L) is incubated with receptor, and the amount of complex R:*L is measured by separating free ligand from the complex. The methods to study receptors rely on a system to form and trap the R:L complex. Great care must be taken to ensure that the label does not interfere with binding. Ligand is mixed with receptor and incubated for a specific period of time and conditions. The R:L complex is trapped by any of several methods, including precipitation, filter binding, separation by magnetic particles, column filtration, and binding to media. The free ligand is washed away or removed by a number of techniques, which usually employ the differences in the size or chemistry of the R:L versus ligand. Filtration is the most common method used as it is rapid and there are numerous options available in the type and specificity of the filtering media. Receptor that has not bound a ligand is not labeled and not detectable under these conditions.
Conditions of the assay are varied so that specific binding is maximized, while nonspecific binding is minimized. The temperature, buffer and ionic conditions, time, and the presence of other components can be tested to optimize the system.
As BS is limited, it can be competed away by an excess of unlabeled ligand. In the presence of the *L and R, an excess (100 × ) of unlabeled L is added, the specific binding is effectively competed for by the unlabeled L, and thus bound *L represents nonspecific binding (except at conditions far below saturating conditions). This is subtracted from the total to obtain the BS (see Fig. 1).
From this data the bound and free *L can be determined, and Scatchard analysis can be performed . The binding affinity (Kd) and number of sites can be determined by plotting bound versus bound/free as shown on the plot. Although plotted differently than enzyme plots, the data obtained are similar in nature, providing both a measure of affinity and a quantitative measure of activity via the number of sites (see Fig. 2). If two types of receptor binding the same ligand are present or if high and low affinity sites are detected, the line will not be straight. A biphasic plot can neatly illustrate the difference between high and low affinity ligands.
Cooperative binding can be determined by using other types of plots. The Hill coefficient is used to determine whether more than one ligand binds and whether there is cooperative behavior [1, 2]. Almost every biochemistry textbook includes a description of oxygen binding by hemoglobin. This example can be used to illustrate cooperative binding of ligand to receptor. Other common examples of cooperative behavior, often found in texts, are binding of transacting factors to DNA. Many of the nuclear hormone receptors, such as estrogen receptor, bind as dimers to DNA responsive elements. Using these examples will help integrate the receptor theory into the course.
A competition curve is a graph used to compare how effectively various ligands bind to a given receptor. This is how various molecules would be tested to design a better drug. If similar drugs are tested to learn more about the affinity of the ligands for the receptor, it is called a structure-activity curve or structure-activity relationship curve. (In all cases, it should be recalled that if a response is not being measured, it is difficult to determine whether the ligand is an agonist or antagonist (see Fig. 3).) A good exam question is to provide competition curve data with ligand structure and chemical data and ask students to interpret the results and design a better drug.
None of this is useful if it is not relevant to the student in context of receptor examples. There are many types of receptors, and these are briefly described in the next section “A Multitude of Receptors.” It is unlikely that all could be covered in detail, but a few examples can be chosen that are consistent with the course focus and interest of the students. For example, sensory receptors are described in the next section; this is an area that many students find fascinating. Recent advances in molecular biology have identified the genes for vision, taste, and smell, and the protein structures of the gene products are being examined and are very exciting. Thus, the biochemistry, molecular biology, signaling events, and receptor theory could all be described about one topic, providing an integrated example of biochemistry research across several subdisciplines. The references included provide information that can be used to obtain specific data to serve as examples when describing the theory of analysis.
A MULTITUDE OF RECEPTORS
Receptors are as plentiful as enzymes. Sometimes a receptor has enzymatic activity. Many enzymes, such as those with allosteric regulation, also qualify as receptors when the broadest use of the term is applied. If we consider any ligand interacting with a macromolecule to be a receptor:ligand interaction, then any interactions of small molecules with protein, DNA, or RNA are types of receptor:ligand complexes if some response occurs. However, for the scope of this article the examples will be limited to those interactions between proteins and small molecules. The number and type is still huge, and defining the interactions in a simple set has not been accomplished. Unlike the IUPAC-IUBMB (International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology) nomenclature for enzymes, which neatly defines enzymes into six categories via function, an established use of receptor classification remains elusive. Receptors serve to sense and respond, communicate, transport molecules, and regulate cellular processes. But there are some categories that can be defined as outlined in the next sections. The IUPHAR (International Union of Pharmacology) has recently developed a system for ongoing nomenclature of receptor subtypes based on structures, functions, and mechanisms (www.iuphar.org/ncpubs.htm).
Within the nervous system, receptors can be classified in a number of ways. Broadly speaking, there are sensory receptors and ligand-activated neuronal receptors. Outside the central nervous system receptors are classified in several ways including transport, signal transduction, and gene regulation. The following sections describe the various physiological properties of sensory, neuronal, and non-neuronal receptors found in organisms.
All sensory systems are designed in ways to allow them to carry out feature detection processes. The manner in which this is accomplished is, in most ways, quite similar across the various sensory modalities. Each sensory system has a set of specialized receptors that only respond to certain kinds of stimuli. Further, these receptors typically respond in a manner that parallels, up to a point, the intensity of that stimulus. Each sensory system contains a multitude of dedicated, line-labeled pathways that carry specific information to the central nervous system. Finally, each sensory system routes information to various distinct cortical areas that are dedicated to the conscious processing of the various features of that sensory modality .
Sensory receptors function to transduce physical energy into a neural code either in the form of generator potentials (excitatory (EPSP) and inhibitory (IPSP) postsynaptic potentials) or action potentials. Sensory receptors are typically classified on the basis of the “sensory” system to which they belong and the stimulus type and range they detect (see Table II). Thus, there are photoreceptors of the visual system that detect photons of light, mechanoreceptors of the auditory and somatosensory systems that respond to mechanical disturbances such as vibration, thermoreceptors of the somatosensory system that detect non-noxious and noxious thermal stimuli, and chemoreceptors of the olfactory, gustatory, and somatosensory system that selectively respond to various exogenous or endogenous chemical agents such as sugar or bradykinin. The type of receptors within each sensory system can also be distinguished on the sensitivity of the receptor to stimulus type, range, and intensity.
Photoreceptors are part of the visual system and respond to a narrow band of electromagnetic energy from 380–760 nm . Photoreceptors are classified as either rods or cones and are so distinguished by both anatomical and physiological properties. The morphology of the outer laminae or outer segment of photoreceptors is more cylindrical for rods and more conical for cones. In terms of physiological response properties, rods not only contain more light-absorbing photopigments than cones, the signal transduction mechanism in rods is greatly amplified. These two characteristics make rods extremely sensitive to light energy such that they are capable of responding to a single photon. Cones, on the other hand, require much higher levels of illumination to carry out their signal transduction processes. It is for these reasons that night and day vision is synonymous with rod and cone vision, respectively. Cones can further be classified by their maximal absorption sensitivities to electromagnetic energy, and this characteristic serves as the basis for color vision. In humans, “blue” cones respond best to short (419 nm) wavelengths of light, “green” cones respond best to medium (531 nm) wavelengths of light, and “red” cones respond best to long (559 nm) wavelengths of light. (The genetic basis for color sensitivity in humans versus other creatures is intriguing to students and a good way to integrate material to gene families.)
The mechanisms of phototransduction in both rods and cones are virtually identical. In the dark, photoreceptors maintain high levels of intracellular cGMP (guanosine 3′:5′-cyclic monophosphate). This, in turn, drives an inward current of positively charged ions that is made up mostly of sodium (Na+) influx through open Na+ channels. This so-called “dark current” produces a somewhat depolarized photoreceptor that results in the release of neurotransmitter that ultimately acts to inhibit retinal bipolar cells downstream (see Fig. 4). The photopigment in photoreceptors (e.g. rhodopsin in rods) contains an opsin portion and the light-absorbing retinal portion. The absorption of light causes the retinal portion to undergo a transformation change from a relatively stable 11-cis-retinal to an unstable all-trans-retinal. As a result, the retinal portion is no longer able to remain bound to the opsin portion, and the two break apart, a process known as the bleaching of the photopigment. The free opsin molecule activates transducin, which leads to elevated levels of cGMP phosphodiesterase. Increasing cGMP phosphodiesterase activity reduces, through a hydrolysis step, cGMP levels, the second messenger responsible for maintaining the dark current. Lowered cGMP levels close Na+ channels and hyperpolarize the photoreceptor. This hyperpolarization decreases the neurotransmitter flux rate (see Fig. 4). Recall that the active release of neurotransmitters in the dark actually inhibits retinal bipolar cells. Light absorption by photopigments causes a biochemical cascade that decreases the inhibitory effects photoreceptors have on bipolar cells. This disinhibition increases the output of bipolar cells to the ganglion cells whose axons form the optic nerve.
Rods and cones are not the only retinal cells that contain light-absorbing opsin molecules. Researchers have recently discovered that a small number of retinal ganglion cells contain melanopsin, a novel light-absorbing opsin molecule. Interestingly, these retinal ganglion cells project their axons to the suprachiasmatic nucleus of the hypothalamus. It has been suggested that these melanopsin-containing ganglion cells are responsible for driving our 24-h sleep-wake cycle called the circadian rhythm.
It is well established that neural communication occurs via electrochemical processes. Signals within neurons take the form of EPSPs, IPSPs, and action potentials mediated by the movement of Na+, potassium (K+), calcium (Ca2+), and chloride through ion channels. Signals between neurons are mediated by the release of neurotransmitters from one neuron that acts on receptors on dendrites and soma of a second or postsynaptic neuron downstream (note that gap junctions are the exception to this rule). Binding of a neurotransmitter at the receptor mediates the influx or efflux of various ions to move through their respective channels to produce EPSPs and IPSPs.
Postsynaptic neural receptors  can be classified by ligand or neurotransmitter binding (e.g. acetylcholine, dopamine, serotonin, etc.) and/or the signal transduction mechanism. For this article, we focus on the latter where postsynaptic neural receptors are classified as either ionophoric or metabotropic. Ionophoric receptors are the simplest in nature and consist of a protein that transverses the phospholipid bilayer of the cell. This protein not only contains a specialized binding site for its respective ligand, it also forms a gated ion channel. Binding of the neurotransmitter to the receptor site causes a conformational change in the entire protein thus opening up the channel to allow for ionic flux. It is interesting to note that each ionophore is highly selective for a given ion; the directional flux of a given ion is determined by both electrostatic forces and concentration gradients. Activation of an ionophoric receptor that is selective for Na+ will produce a depolarized (EPSP) cell via Na+ influx. Activation of an ionophoric receptor that is selective for K+ will produce a hyperpolarized (IPSP) cell via K+ efflux (see Fig. 5).
Neural communication via metabotropic receptors is also known as second messenger systems. In contrast to ionophoric receptors, metabotropic receptors do not form ion channels. Rather the receptor is functionally linked to a regulatory G-protein that initiates one of several intracellular biochemical cascades that ultimately leads to an EPSP or IPSP. As before, the process begins with the binding of a neurotransmitter to the receptor. This activates a GTP-binding protein that stimulates adenylyl cyclase that converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). cAMP, serving as the second messenger in this system (the neurotransmitter is considered the first messenger), acts either directly or indirectly through various protein kinases to modulate the opening and/or closing of ion channels (see Fig. 5). Other second messengers include cGMP, diacylglycerol, and inositol phosphate systems. As one might expect, a second messenger that ultimately opens Na+ channels results in an EPSP, while a second messenger that ultimately opens K+ channels results in an IPSP. There is one important signal transduction difference between ionophoric receptors and metabotropic receptors. Activity at a metabotropic receptor yields a much greater postsynaptic response than that found at ionophoric receptors. This is due to the amplification of the signal through the intracellular biochemical cascade in which many more ion channels are affected.
Other kinds of ligand-activated neuronal receptors are found at presynaptic sites on terminal buttons where they serve various other functions. Some receptor proteins are autoreceptors whose function is to monitor overall synaptic activity and to provide feedback to the presynaptic neuron to regulate the synthesis and release of neurotransmitters. Too much or too little synaptic activity, measured in terms of ligand binding at the autoreceptor, results in down- or up-regulation, respectively, of these processes. Another set of ligand-binding proteins called transporter proteins act to take up unused neurotransmitters from the synaptic cleft. These neurotransmitters are repackaged in vesicles for later use. This form of reuptake is an important mechanism for termination of synaptic activity and is a target of modulation by a number of psychotherapeutics including the antidepressant drug Prozac, a serotonin reuptake inhibitor.
Examples of non-neuronal receptors include receptors involved in gene activation and transport systems. Receptors that regulate gene expression, including those that bind ligands as part of a gene regulatory process, are described in texts. A common example is the lac operon induction and repression system. Although included as a gene regulatory system, it should be recognized and reinforced as receptor:ligand complexes of the lac repressor, inducer, and DNA. Other models are included in textbook sections on transacting factors, such as the hormone receptor superfamily.
Transport of molecules is critical to cell communication and metabolism. It can include intercellular and interorgan trafficking as well as internalization and intracellular trafficking of receptor:ligand complexes. In introductory biology, students learn the broad categories and mechanisms of transport at the cell surface, including passive and active transport, diffusion, and phagocytosis. In studying metabolism, references are briefly made to transporters that move molecules across membranes. Details of transport systems within cells and between organs are less commonly understood.
A classic example can be found by studying the experiments on cholesterol and low density lipoprotein (LDL) transport that won Brown and Goldstein  the Nobel Prize in 1985 (www.nobel.se/medicine/laureates/1985/brown-goldstein-lecture.pdf). They studied the transport system in patients that suffered from familial hypercholesterolemia, a disease causing heart attacks and other health defects in family members and severe enough to cause heart attacks in small children. They found that the patients had a mutant form of the LDL receptor and could not properly transport the LDL complex, which carries cholesterol. As a result, dangerous levels of cholesterol were present in the blood of the patients, and feedback information to the enzymes controlling cholesterol synthesis was interrupted. Their work on the receptor led to understanding of how molecules bind to surface receptors and are internalized into organelles within the cytoplasm. The researchers coined the term “receptor-mediated endocytosis” to describe the internalization. They also discovered the genetic cause for the disease, a mutation in the LDL receptor gene that caused truncation in the protein-coding region of the receptor. This loss caused the receptor to be unable to properly migrate on the cell surface to a site where it could be internalized by clathrin-coated vesicles.
REGULATION OF RECEPTORS
An important area of study is the regulation and plasticity of receptors with research on the neurophysiological substrates of learning and memory showing explosive growth in the last decade. However, the notion that plastic changes in the nervous system mediated by the dynamic regulation of neurons and receptors was first articulated more than one-half century ago. Donald Hebb  theorized that information processed in the nervous system could lead to repetitive activity in certain neural circuits. This sustained pattern of activation could, in turn, induce alterations in the number and structure of synaptic junctions making these connections stronger and more efficacious and rendering them much more likely to fire in the future. Hebb argued that functional modifications at the synapse, produced by reverberating circuits, could serve as the basis of learning in the nervous system. Memories, then, are simply the reactivation of these modified neural circuits through either internal or external stimuli.
In the early 1970s, working with anesthetized rabbits, Bliss and Lømo  reported that high frequency stimulation of the perforant path led to enhanced postsynaptic responses in the dentate gyrus, a phenomenon now referred to as long term potentiation, or LTP for short. Neurons that form the perforant path lead to enhanced postsynaptic responses in cells of dentate gyrus. This phenomenon is referred to as LTP. Considerable research from the last 3 decades has detailed many of the basic neurochemical mechanisms responsible for the induction, maintenance, and expression of LTP . It is well established that LTP requires the involvement of the excitatory amino acid neurotransmitter glutamate, its N-methyl-D-aspartate (NMDA) receptor, and the influx of Ca2+ after the release of the magnesium (Mg2+) block through the channel of this receptor. Some models indicate glutamate acting on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors causes an initial depolarization (EPSP) that is necessary for the displacement of Mg2+ from the NMDA ion channel (however, any input source capable of producing an EPSP can produce a similar result). Calcium entry into the cell through the NMDA channel leads to the subsequent activation of numerous Ca2+-dependent enzymes including protein kinase C (PKC), Ca2+-calmodulin kinase II (CaMKII), and nitric-oxide synthase (NOS). The action of CaMKII is to phosphorylate AMPA receptor subunits that render previously non-functional AMPA receptors functional. In addition, the retrograde action of nitric oxide (NO) leads to an increase in glutamate release from presynaptic terminals (see Fig. 6). In sum, activity-dependent LTP is mediated by extensive modification of synaptic processes with more glutamate being released presynaptically and up-regulated AMPA receptors found postsynaptically (metabotropic glutamate receptors have also been implicated in plasticity).
LTP is not the only form of plasticity in the nervous system. Dendritic branching, spine modification, long term depression (i.e. activity-dependent decrease in excitability of neurons), and depotentiation (i.e. reversal of an LTP event) are other examples of the dynamic changes that occur in the central nervous system. And these forms of plasticity, along with LTP itself, involve many other neurotransmitter systems, hormones, nerve growth factors, and enzymatic processes than those previously discussed . Plasticity in the nervous system has been identified nearly everywhere researchers look including brainstem, cerebellum, amygdala, septum, hippocampus, and sensory, motor, and association cortical areas.
Receptor biochemistry is crucial to understanding a large segment of the field. This information is not just critical to understanding biochemistry. It is necessary theory for any biochemist studying the metabolic processes and cellular communication. As many as half of the drugs on the market are based on some interaction, activation, or inhibition of receptor proteins. Many students will enter other fields, becoming chemists, immunologists, pharmacologists, or other professionals that require an understanding and practical knowledge of receptor theory and technique, or become members of teams that explore receptor control and drug design. Students will be better prepared for graduate school or the job market if they have an understanding of the logic and tools of the receptor field.
In our classes, students always respond positively to this material as it is exciting, relevant, and critical to knowing how to solve the problems they will tackle in medicine, psychology, pharmacy, agriculture, research, and other fields. We hope this article encourages other faculty to include more of this material in their lectures.
Table Table I. Comparison of terms and events in enzymatic and receptor processes
What is bound
The complex formed
How to measure the event
Measure product formed
Measure R:L complex formation or a response
Number of binding sites
One or more sites
One or more sites
Type of binding
Specific or general
Specific or general
Measurement of binding affinity
Molecules that also bind to protein
Allosteric activation, gene regulation
Allosteric activation, gene regulation
Table Table II. Sensory systems and their receptors
Photons of light
Rods and cones
Changes in air pressure
Body orientation and motion
Hair cells and others
Mechanical, thermal, and chemical
Mechano-, thermo-, and chemoreceptors
Pacinian, Ruffini, and Meissner corpuscles, Merkels disks and nociceptors
The abbreviations used are: E, enzyme; S, substrate; R, receptor; L, ligand; BS, specific binding; BN, nonspecific binding; BT, total binding; *L, radioactive ligand; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; LDL, low density lipoprotein; LTP, long term potentiation; NMDA, N-methyl-D-aspartate; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; PKC, protein kinase C; CaMKII, Ca2+-calmodulin kinase II; NOS, nitric-oxide synthase.