Cell surface receptor

Membrane receptors are specialized protein molecules in the membranes of cells, to which extracellular signal molecules (usually hormones or cell recognition molecules) attach, triggering changes in the function of the cell. This process is called signal transduction: the external signal is transduced into intracellular action. They are hundreds of receptors are known and there are undoubtedly many more yet to be discovered.

Almost all known membrane receptors are proteins. They can be various kinds of proteins such as glycoprotein, lipoprotein, glycolipidprotein, etc. A certain cell membrane can have several membrane receptors with various amounts on its surface. A certain receptor may also have different concentrations on different membrane surfaces, depending on the membrane and cell function. The receptors usually form “clusters” on the membrane surface, therefore the distribution of receptors on membrane surface is mostly heterogeneous.

Structure (in general)

Generally, all kinds of membrane receptors consist of three domains:

• ligand-binding domain: Basically, a ligand-binding domain faces the exterior of a cell. This domain will bind with extracellular ligand. Cell signals would be transduced through the whole membrane receptor into the cytoplasm.

• cytoplasmic(effector) domain: This domain faces the interior of a cell. This domain will be activated by conformation changing or by binding with particular intracellular proteins. Thus, it can interact with further proteins, and so on, finally leading to a cellular response to that extracellular signal.

• transmembrane domain: This domain is embedded in the membrane. The conformation change caused by ligand-binding domain binding with extracellular ligand would be transmitted to the this domain and passed to the effector(coupling) domain to activate intracellular response.

Categories

Based on the structural and functional similarity, membrane receptors are mainly divided into 3 classes: The ion channel-linked receptor; The enzyme-linked receptor and G-protein-linked receptor.

• Ion channel-linked receptors: These receptors are ion-channels (including cation-channels and anion-channels) themselves and constitute a large family of multipass transmembrane proteins. They are involved in rapid signaling events most generally found in electrically excitable cells such as neurons and are also called ligand-gated channels. Opening and closing of Ion channels are controlled by neurotransmitters.

• Enzyme-linked receptors: These receptors are either enzymes themselves, or are directly associated with the enzymes that they activate. These are usually single-pass transmembrane receptors, with the enzymatic portion of the receptor being intracellular. The majority of enzyme-lined receptors are protein kinases, or associate with protein kinases.

• G protein-linked receptors: G protein-linked receptors are integral membrane proteins that possess seven membrane-spanning domains or transmembrane helices. These are receptors that, upon ligand binding, activate a G protein. G-protein is a trimeric GTP-binding regulatory protein which has 3 subunits: α、β and γ. The α subunit can bind with GDP. The binding of the ligand to the receptor would cause the phosphorylation of GDP and activate the α subunit, which will then dissociate with β and γ subunits. The activated α subunit of G protein can further affect other intracellular signaling proteins, or target proteins directly.

Signal Transduction

The External Reactions and the Internal Reactions for signal transduction

Signal transduction process through membrane receptors involve the External Reactions in which the ligand binds to a membrane receptor and the Internal Reactions in which intracellular response is triggered.

Signal transduction through membrane receptors usually requires 4 characters: 1. Extracellular signal molecule: an extracellular signal molecule is produced by one cell and is capable of traveling to neighboring cells, or to cells that may be far away. 2. Receptor protein: the cells in an organism must have cell surface receptor proteins that bind to the signal molecule and communicate its presence inward into the cell. 3. Intracellular signaling proteins: these distribute the signal to the appropriate parts of the cell. The binding of the signal molecule to the receptor protein will activate intracellular signaling proteins that initiate a signaling cascade (a series of intracellular signaling molecules that act sequentially). 4. Target proteins: the conformations or other properties of the target proteins are altered when a signaling pathway is active and changes the behavior of the cell.

Ion Channel-Linked Receptor

Three conformation states of acetylcholine receptor

In the signal transduction event in a neuron, the neurotransmitter binds with the receptor and alters the conformation of the protein, which opens the ion-channel, allowing extracellular ions go into the cell. The ion permeability of the plasma membrane is altered, and this will instantaneously convert the extracellular chemical signal into intracellular electric signal, which will alter the excitability of the cell.

Acetylcholine receptor is a kind of cation-channel linked receptor. The protein consists of 4 subunits: α, β, γ, and δ subunits. There are two α subunits, containing one acetylcholine binding site each. This receptor can exist in three different conformations. The unoccupied-closed state is the protein at its original conformation. After two molecules of acetylcholine bind simultaneously to the binding sites on α subunits, the conformation of the receptor is altered and the gate is opened, allowing for the penetration of many ions and small molecules. However, this occupied-open state can only last for a very short period of time and then the gate is closed again, forming the occupied-closed state. The two molecules of acetylcholine will quickly dissociate from the receptor and the receptor will returns to its unoccupied-closed state and is ready for next transduction cycle again.

Enzyme-Linked Receptors

example sketch of an enzyme-linked receptor structure (structure of IGF-1R)

Currently there are 6 known types of enzyme-linked receptors: Receptor tyrosine kinases; Tyrosine kinases associated receptors; Receptor-like tyrosine phosphatases; Receptor serine/threonine kinases; Receptor Guanylyl cyclases and Histidine kinase associated receptors. Receptor tyrosine kinases is the one kind with the largest population and most widely application. The majority of these molecules are receptors for growth factors and hormones like epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor(HGF), insulin, nerve growth factor (NGF) etc.

Most of these receptors will dimerize after binding with their ligands in order to activate further signal transductions. For example, after the epidermal growth factor (EGF) receptor binds with its ligand EGF, two receptors dimerize and then undergo phosphorylation of the tyrosine residues in the enzyme portion of each receptor molecule, which will activate the tyrosine protein kinase and analyze further intracellular reactions.

G Protein-Linked Receptors

G protein-linked receptor mechanism

Dissociation/association of the G protein

There are two principle signal transduction pathways involving the G-protein linked receptors: cAMP signal pathway and Phosphatidylinositol signal pathway.

cAMP signal pathway The cAMP signal transduction contains 5 main characters: stimulative hormone receptor (Rs) or inhibitory hormone receptor (Ri)；Stimulative regulative G-protein (Gs) or inhibitory regulative G-protein (Gi)；Adenylyl cyclase; Protein Kinase A (PKA); and cAMP phosphodiesterase.

Activation effects of cAMP on Protein Kinase A

The effect of Rs and Gs in cAMP signal pathway

The effect of Ri and Gs in cAMP signal pathway

Stimulative hormone receptor (Rs) is a receptor that can bind with stimulative signal molecules, while inhibitory hormone (Ri) is a receptor that can bind with inhibitory signal molecules.

Stimulative regulative G-protein is a G-protein linked to stimulative hormone receptor (Rs) and its α subunit upon activation could stimulate the activity of an enzyme or other intracellular metabolism. On the contrary, inhibitory regulative G-protein is a linked to an inhibitory hormone receptor and its α subunit upon activation could inhibit the activity of an enzyme or other intracellular metabolism.

The Adenylyl cyclase is a 12 transmembrane glucoprotein that catalyzes ATP to form cAMP with the help of cofactor Mg2+or Mn2+. The cAMP produced is a second messenger in cellular metabolism and is an allosteric activator to Protein kinase A.

Protein kinase A is an important enzyme in cell metabolism due to its ability to regulate cell metabolism by phosphorylating specific committed enzyme in matabolic pathway and it can also regulate specific gene expression, cellular secretion and membrane permeability. The protein enzyme contains two catalytic subunits and two regulative subunits. When there is no cAMP，the complex is inactive. After cAMP binds with the regulative subunits, it alters the conformation of these subunits, causing the dissociation of the regulative subunits, which activate protein kinase A and allow for further biological effects.

cAMP phosphodiesterase is an enzyme that can degrade cAMP to 5’-AMP, which will terminate the signal.

Phosphatidylinositol signal pathway In phosphatidylinositol signal pathway the extracellular signal molecule binds with the G-protein receptor on cell surface and active phospholipase C which is located on the plasma membrane. The lipase hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messegers: Inositol 1,4,5-triphosphate (IP3) and Diacylglycerol (DAG). IP3 binds with the receptor in the membrane of the smooth endoplasmic reticulum and mitochondria, help open the Ca2⁺ channel and DAG will help activate Protein Kinase C (PKC), which will cause series of biological effects. DAG will help activate Protein Kinase C, which phosphorylates many other proteins, changing their catalytic activities, leading to cellular responses. The effects of Ca2⁺ is also remarkable: it cooperates with DAG in activating PKC and can activate CaM kinase pathway, in which calcium modulated protein calmodulin (CaM) binds Ca2⁺, undergoes a change in conformation, and activates CaM kinase II, which has unique ability to increase its binding affinity to CaM by autophosphorylation, making CaM unavailable for the activation of other enzymes. The kinase then phosphorylates target enzymes, regulating their activities. The two signal pathways are connected together by Ca2⁺-CaM, which is also a regulatory subunit of adenylyl cyclase and phosphodiesterase in cAMP signal pathway.

Structure-Based Drug Design for Membrane Receptors

Flow charts of two strategies of structure-based drug design

As the experimental methods as X-ray crystallography and NMR develop, the amount of information concerning 3D structures of biomolecular targets has increased dramatically, as well as the structural dynamic and electronic information about the ligands. This encourages the rapid development of the structure-based drug design. Current methods for structure-based drug design can be divided roughly into two categories. The first category is about “finding” ligands for a given receptor, which is usually referred as database searching. In this case, a large number of potential ligand molecules are screened to find those fitting the binding pocket of the receptor. This method is usually referred as ligand-based drug design. The key advantage of database searching is that it saves synthetic effort to obtain new lead compounds. Another category of structure-based drug design methods is about “building” ligands, which is usually referred as receptor-based drug design. In this case, ligand molecules are built up within the constraints of the binding pocket by assembling small pieces in a stepwise manner. These pieces can be either atoms or fragments. The key advantage of such a method is that novel structures, not contained in any database, can be suggested. These techniques are raising much excitement to the drug design community.

Active sight identification

Active sight identification is the first step in this program. It analyzes the protein to find the binding pocket, derives key interaction sites within the binding pocket, and then prepares the necessary data for Ligand fragment link. The basic inputs for this step are the 3D structure of the protein and a pre-docked ligand in PDB format, as well as their atomic properties. Both ligand and protein atoms need to be classified and their atomic properties should be defined, basically, into four atomic types:

• hydrophobic atom: all carbons in hydrocarbon chains or in aromatic groups.
• H-bond donor: Oxygen and nitrogen atoms bonded to hydrogen atom(s).
• H-bond acceptor: Oxygen and sp2 or sp hybridized nitrogen atoms with lone electron pair(s).
• Polar atom: Oxygen and nitrogen atoms that are neither H-bond donor nor H-bond acceptor, sulfur, phosphorus, halogen, metal and carbon atoms bonded to hetero-atom(s).

The space inside the ligand binding region would be studied with virtual probe atoms of the four types above so the chemical environment of all spots in the ligand binding region can be known. Hence we are clear what kind of chemical fragments can be put into their corresponding spots in the ligand binding region of the receptor.

Ligand fragment link

Flow chart for structure based drug design

When we want to plant “seeds” into different regions defined by the previous section, we need a fragments database to choose fragments from. The term “fragment” is used here to describe the building blocks used in the construction process. The rationale of this algorithm lies in the fact that organic structures can be decomposed into basic chemical fragments. Although the diversity of organic structures is infinite, the number of basic fragments is rather limited.

Before we put the first fragment, i.e. seed, into the binding pocket, and add other fragments one by one. we should think some problems. First, the possibility for the fragment combinations is huge. A small perturbation of the previous fragment conformation would cause great difference in the following construction process. At the same time, in order to find the lowest binding energy on the Potential energy surface (PES) between planted fragments and receptor pocket, the scoring function calculation would be done for every step of conformation change of the fragments derived from every type of possible fragments combination. Since this requires a large amount of computation, one may think using other possible strategies to let the program works more efficiently. When a ligand is inserted into the pocket site of a receptor, conformation favor for these groups on the ligand that can bind tightly with receptor should be taken priority. Therefore it allows us to put several seeds at the same time into the regions that have significant interactions with the seeds and adjust their favorite conformation first, and then connect those seeds into a continuous ligand in a manner that make the rest part of the ligand having the lowest energy. The conformations of the pre-placed seeds ensuring the binding affinity decide the manner that ligand would be grown. This strategy reduces calculation burden for the fragment construction efficiently. On the other hand, it reduces the possibility of the combination of fragments, which reduces the number of possible ligands that can be derived from the program. These two strategies above are well used in most structure-based drug design programs. They are described as “Grow” and “Link”. The two strategies are always combined in order to make the construction result more reliable.

Scoring method

Structure-based drug design attempts to use the structure of proteins as a basis for designing new ligands by applying accepted principles of molecular recognition. The basic assumption underlying structure-based drug design is that a good ligand molecule should bind tightly to its target. Thus, one of the most important principles for designing or obtaining potential new ligands is to predict the binding affinity of a certain ligand to its target and use it as a criterion for selection.

A breakthrough work was done by Bohm to develop a general-purposed empirical function in order to describe the binding energy. The concept of the “Master Equation” was raised. The basic idea is that the overall binding free energy can be decomposed into independent components which are known to be important for the binding process. Each component reflects a certain kind of free energy alteration during the binding process between a ligand and its target receptor. The Master Equation is the linear combination of these components. According to Gibbs free energy equation, the relation between dissociation equilibrium constant, Kd and the components of free energy alternation was built.

File:Master Equation in Scoring Function.jpg

The sub models of empirical functions differ due to the consideration of researchers. It has long been a scientific challenge to design the sub models. Depend on the modification of them, the empirical scoring function is improved and continuously consummated. Commonly, the following facets are identified of a good scoring function.