PArrestin bindingmediated GPCR endocytosis

P-Arrestins act as endocytotic adaptor proteins targeting GPCRs to clathrin-coated pits (Figure 123, Figure 124). To this end, they bind with high affinity to clathrin as well as to the p2-adaptin subunit of the heterotetrameric AP-2 adaptor complex. When present in the clathrin-coated pits, the GPCRs may be internalized (also called sequestered) within the cell by an endocytotic process. This process is dependent on dynamin, a large GTPase that is involved in the pinching off of...

Regional distribution of receptors

Radioligand binding on membrane preparations provides information concerning the interaction between drugs and well-defined receptors, as well as about the occurrence of such receptors in certain tissues or organs. However, most tissues and organs are very complex and comprise a number of different cell types, each with their own receptor and response specificity. In this context, the brain is especially complex since it contains a great number of different neurones. Yet, neurones that are...

From receptor to response introduction to GPCRs

All drugs that are presently on the market for clinical therapy are estimated to target less than 500 biomolecules, ranging from nucleic acids to enzymes, G protein-coupled receptors (GPCRs) and ion channels (Figure 71). Presently, GPCRs constitute one of the principal targets of drugs used in pharmacology and more than 1000 genes encoding GPCRs have been identified from human genome sequencing efforts. This represents a substantial part ( 3 ) of the human genome. A great deal of information...

The general allosteric ternary complex model

The term 'allosteric' (from the Greek meaning 'other site') was introduced by Monod et al. (1965) to define binding of ligands to sites on enzymes that were topographically distinct from the substrate-binding site. These accessory binding sites were called 'allosteric sites', in contrast to the substrate-binding (active) site, which was defined as the 'isosteric site'. For GPCRs, the orthosteric site refers to the agonist binding Figure 213 Allosteric models for the binding of two ligands (A...

Multiple receptor states related to truncation covalent modification and mutation

In addition to the ligand-mediated changes in receptor conformation, processes like covalent modification, truncation and mutation are also prone to affect the way in which receptors interact with other molecules. As an example of covalent modification, the phosphorylated receptor clearly represents another 'state' to the 'activated' receptor. Accordingly, 'activated' and Figure 207 Left Dissociation of 3H candesartan from wild-type AT1 receptors (negative charge, polar Lys199) or mutated...

Multistate receptors and multiple ligand binding sites

In the multistate model (Schwartz et al., 1995), the receptor is proposed to alternate spontaneously between multiple active and inactive conformations. The key elements in this model are The biological response to a given ligand is determined by the receptor conformation to which this ligand binds with highest affinity. If the preferred conformation is recognized by the G protein as active, the ligand will behave like an agonist. If the preferred conformation is inactive, the ligand will...

Agonist trafficking what do models predict

In the initial model by Kenakin, there is a limitless number of active receptor conformations. For simulation studies, this was simplified by Leff et al. (1997) to three receptor conformations with one inactive (R) and two active conformations (R* and R**). In the intact three-state model (Figure 198), it was further assumed that analysis with antagonists 3 receptor types analysis with antagonists 3 receptor types analysis with agonists 9 receptor types Figure 197 Schematic diagram of three...

Early models for GPCR activation

Earlier mechanistic hypotheses largely emphasised the fluid-mosaic model of plasma membrane structure (Singer and Nicolson, 1972) and the notion of 'collision coupling' of the components. Implicit in this explanation was the idea that GPCRs, G proteins and effector components were physically separate entities that were free to diffuse in the membrane. Models that gathered widespread acceptance in the late seventies and eighties are the Collision Coupling Model (Tolkovsky and Levitzki, 1978) and...

Adrenergic Drugs Preface

This book provides, in the first place, useful scientific background to those involved in pre-clinical research and to those who need to function within multi-disciplinary teams in the pharmaceutical industry (from medical chemistry and molecular biology via pharmacology to drug marketing). Today's education of researchers in the pharmaceutical industry needs to be broad and insight into pharmacological issues needs to be warranted. In this respect, the focus on G protein-coupled receptors...

Kinetic experiments

Unlike the saturation and competition binding experiments, kinetic studies provide information about the time-course of the binding. These studies usually comprise two types of experiments (Figure 45) Determination of dissociation rate constant in these experiments the radioligand is incubated with the receptor and the dissociation is initiated either by adding an excess of unlabelled ligand (so that free receptors are immediately occupied and no longer accessible to the radioligand) or by...

Mechanisms to terminate signalling

Cellular responses to agonists of the GPCRs are usually rapidly attenuated. Signals may be attenuated by mechanisms that operate at the level of the agonist, the receptor, the G proteins and at numerous downstream steps of the signalling pathway. Several processes contribute to the removal of hormones and neurotransmitters from the extracellular fluid, but their relative importance depends on the nature of the agonist (Figure 130) For all agonists, dilution in the extracellular fluid and...

Chemical messengers and the cell membrane

Higher organisms are composed of a multitude of cell types each possessing specialized physiological functions. Their harmonious co-existence is only possible if they can communicate with each other i.e. if they can exchange information concerning their respective needs. Cell signalling can result either from direct interaction of a cell with its neighbour (juxtacrine signalling) or from the exchange of small molecules, i.e. 'chemical messengers'. These chemical messengers will only induce...

Paracrine signalling by local chemical messengers

Local chemical messengers are only active in the vicinity of the cells from which they are secreted. Extracellular enzymes rapidly destroy many of them or they are even very unstable of their own. Hence, they are quickly transformed into inactive metabolites so that they can only diffuse over a short distance. Some cells respond to signalling molecules that they themselves produce (autocrine signalling). In this respect, it is noteworthy that abnormal autocrine signalling frequently contributes...

Allosteric phenomena at GPCR detection by radioligand binding

Allosteric phenomena at GPCRs can be evidenced using radioligand binding and functional assays. Usually, they are first detected when experimental data deviate from the expectations of simple (competitive) mass-action kinetics. Yet such findings may also reflect experimental artefacts, including inappropriate drug equilibration times, drug solubility problems, exceedingly high receptor concentrations or perturbation of the surrounding lipid bilayer. This latter mechanism allows many types of...

Techniques to study G proteincoupling preference

Several experimental strategies have been employed to analyze the selectivity of receptor-G protein interactions Transient co-expression of individual GPCRs with different Ga subunits in cultured mammalian cells. Reconstitution of purified receptors and G protein subunits in artificial lipid bilayers. This represents a very difficult and laborious task. Moreover, receptor-G protein interactions are studied in a highly artificial environment. 'Immuno-neutralization' studies have been performed...

Saturation binding

These experiments provide information about the concentration of a receptor. They are solicited to compare the concentrations of different receptors in a given tissue and to monitor variations in receptor concentration as a result of normal physiological regulation, medication and pathophysiological conditions. For saturation binding experiments, constant amounts of membrane suspension are incubated with increasing concentrations of radioligand. Obviously, both total and non-specific binding...

Endocrine signalling by hormones

Image Glucagon Preprohormone Structure

Endocrine glands (or nerve endings for neurohormones) secrete hormones into the blood stream. They can be transported to almost any part of the body. For the hormones, receptors may thus be located in or on cells from distant tissues. Typical examples are adrenaline which is secreted by the chromaffin cells of the adrenal medulla and which acts on a great number of tissues in the body and insulin which is secreted by the pancreatic P-cells and which acts on the liver and the adipose tissue....

Technical aspects of radioligand binding

Radioligand Binding Assay Principle

For a long time, hormone and neurotransmitter receptors remained abstract concepts whose existence was proposed only to explain pharmacological effects on target tissues. Since the mid-seventies, it has become possible to investigate of the receptor molecules themselves by the means of radioligand binding. This technique also allows the direct evaluation of the binding properties of any compound for a given membrane-bound receptor. Very often, radioligand binding experiments are performed with...

Introductory comments

GPCRs are traditionally known to behave as monomers. Yet, there is increasing evidence that they form dimers and physically interact with a variety of other membrane proteins. These include other receptors as well as non-receptor membrane proteins that affect GPCR cell surface expression, binding and functional properties Figure 139 . In certain situations, this has clearly been shown to alter the pharmacological profile of the GPCR. Figure 139 GPCR dimerization and association with chaperones...

Doseresponse curves and associated problems

Stimulation of a membrane-bound receptor by an agonist will provoke the onset of a whole chain of intracellular events. These events will ultimately lead to a 'physiological' response. This response, as well as intermediate intracellular events, can be measured to obtain indirect information about the receptor, for example, to investigate the effect of P-adrenergic drugs on the heart. One of the most proximate 'biochemical' responses is adenylate cyclase stimulation. The activity of this enzyme...

Inverse agonism

In contrast to agonists that produce receptor activation and neutral antagonists that do not affect basal receptor activity, inverse agonists are able to decrease basal receptor Figure 177 The cubic ternary complex model Weiss et al., 1996 allows the inactive receptor state to interact with a G protein circled . Reprinted from Journal of Theoretical Biology, 181, Weiss, J. M., Morgan, P. H., Lutz, M. W. amp Kenakin, T. P., The cubic ternary complex receptor-occupancy model III. Resurrecting...

Receptor classification and antagonist affinity

Adrenergic Receptors

Adrenergic receptors were initially classified in 1948 by Ahlquist into the a- and P-subtypes on basis of differences in the order of potencies of agonists Figure 61 The a-adrenergic receptor is associated with most of the excitatory functions e.g. vasoconstriction and contraction of the smooth muscle of the uterus . The potency of catecholamines to trigger these responses decreases in the order adrenaline gt noradrenaline gt gt isoproterenol. The P-adrenergic receptor is associated with most...

The nervous system and synaptic signalling by neurotransmitters

Autonomic Nervous System Divisions

The nervous system has two divisions The central nervous system CNS consists of the brain and spinal cord. The peripheral nervous system consists of cranial and spinal nerves. It includes the autonomic nervous system. The peripheral nervous system relays information to and from the central nervous system. The brain is the centre of activity that integrates this information and initiates responses. The autonomic nervous system part of the peripheral nervous system has two divisions sympathetic...

Competition binding

Radioligands are fairly expensive and only very few of them are specific enough for the purpose of receptor identification. Fortunately, radiolabelling of a drug is not strictly required for determining its affinity for a given receptor. This parameter can indeed be determined on basis of the drug's ability to compete with a specific radioligand for binding to that receptor. These competition binding experiments are now widely used by pharmacologists as a screening tool to evaluate the affinity...

Membrane proteins and membrane receptors

Receptor Proteins Cell Membrane

Membrane proteins have diverse functions. Some of them consolidate the structure of the membrane, whereas others have more active functions, such as recognition hormone, neurotransmitter and antibody receptors , pumping ATPases , regulation of the inflow of ions and metabolites calcium-gate, glucose channel and enzymatic functions phospholipases, adenylate cyclase . With regard to their degree of incorporation into the plasma membrane, proteins can be divided into two categories Figure 22 The...

GPCR structure

7tm Receptor

During the mid-seventies, it became possible to identify the P-adrenergic receptors directly i.e. by radioligand binding and soon afterwards the receptors could be purified by affinity chromatography. Amino acid sequences from small fractions of the purified receptors could be determined, and this opened new horizons for the molecular biologist. Figure 80 A Three-dimensional structure of bacteriorhodopsin reprinted from Biochimica Biophysica Acta, 1460, Subramaniam S. and Henderson R....

Antagonist preincubation no receptor reserve

Non Competitive Antagonist Agonist

Functional studies very often include a pre-incubation step, in which the receptors are pre- equilibrated with the antagonist. Subsequently, the agonist will be added and the response measured. This implies that the antagonist had the opportunity to interact with the receptor for some time without any interference from the agonist. Therefore, one prefers to speak in terms of surmountable parallel shifts of the dose-response curves and insurmountable antagonism depression of the maximal response...

State

Studies with a2adrenergic, and many other GPCRs, indicate that they are capable of activating different types of G proteins. According to a two-state receptor model i.e. where the receptor can only reside in an inactive or an active conformation , the role of agonists is to increase the number of activated receptors. Each agonist will have its own intrinsic efficacy e for a given type of receptor Figure 181 . Hence, the amount of activated receptors which may be regarded to represent the...

Coincubation no receptor reserve

Non Competitive Antagonism

A rightward shift of the dose-response curves Figure 224 without decrease in the maximal response can be observed for competitive antagonists Figure 226 . They compete for binding to the same or to partially overlapping sites at the receptor so that the binding of the one excludes the binding of the other. They produce a rightward shift of the agonist dose-response curve without affecting the maximal response. The shift increases with the antagonist concentration and is, in principle,...

Hydrophobicity effect on release and transport of messengers

Cell Membrane Hydrophobic Effect

The cell membrane constitutes a hydrophobic barrier between the cell and its environment. It is constituted of a double layer of lipids mainly phospholipids but also Figure 14 Synthesis and action of NO . Figure 14 Synthesis and action of NO . glycolipids and cholesterol wherein proteins are able to 'float'. The membrane lipids Figure 15 are amphitatic molecules i.e. they possess a hydrophobic and a hydrophilic part. For the phospholipids, the hydrophobic part is constituted of two long up to...

Membrane compartimentalization

Bradykinin Receptor Cyclase

Controversy has arisen over why agonist competition curves are shallow in the first place. The initial explanation was based on the assumption that there is a stoichio-metric limitation to the amount of G proteins. However, direct methods of measurement have subsequently shown that G proteins may be present in large excess over the number of cognate receptor proteins. For example, there is a substantial excess of Gs relative to both P-adrenergic receptors and adenylate cyclase in S49 murine...

GPCR dimerization

Principle Luciferase Gfp Renilla

GPCRs are traditionally regarded to exist and to be fully functional as monomers. Yet, recent findings suggest that they also exist as homo- as well as heterodimers i.e. dimers between, respectively, the same and different receptor molecules . As dimerization is often observed in recombinant cell systems, there is a major concern that this process could be due to receptor overexpression and, hence, about the physiological relevance of this process. However, dimerization has also been observed...