Dopamine and its receptor

Dopamine and its receptor

The neurotransmitter dopamine is the primary endogenous ligand for dopamine receptors. Dopamine receptors are a class of metabotropic G protein-coupled receptors that are prominent in the vertebrate central nervous system (CNS).

Biosynthesis of Dopamine

Dopamine, a catecholamine, is synthesized in the terminals of dopaminergic neurons from tyrosine, which is transported across the blood-brain barrier by an active process. The rate-limiting step in the synthesis of dopamine is the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA), catalyzed by the enzyme tyrosine hydroxylase. L-DOPA is converted rapidly to dopamine by aromatic L-amino acid decarboxylase.

Fig. 1 Dopaminergic terminal.

Location

CNS- Assays of distinct regions of the CNS eventually revealed that the distributions of dopamine and norepinephrine are markedly different. In fact, more than half the CNS content of catecholamine is dopamine and extremely large amounts are found in the basal ganglia, the nucleus accumbens, the olfactory tubercle, the central nucleus of the amygdala, the median eminence, and restricted fields of the frontal cortex.

Cardio-pulmonary- In humans, the pulmonary artery expresses D1, D2, D4, and D5 and receptor subtypes, which may account for vasorelaxive effects of dopamine in the blood. In rats, D1-like receptors are present on the smooth muscle of the blood vessels in most major organs.

D4 receptors have been identified in the atria of rat and human hearts. Dopamine increases myocardial contractility and cardiac output, without changing heart rate, by signaling through dopamine receptors.

Renal- Dopamine receptors are present along the nephron in the kidney, with proximal tubule epithelial cells showing the highest density. In rats, D1-like receptors are present on the juxtaglomerular apparatus and on renal tubules, while D2-like receptors are present on the renal tubules, glomeruli, postganglionic sympathetic nerve terminals, and zona glomerulosa cells of the renal cortex. Dopamine signaling affects diuresis and natriuresis.

Storage and release

In dopaminergic nerve terminals, dopamine is taken up into vesicles by a transporter protein; this process is blocked by reserpine, which leads to depletion of dopamine. Release of dopamine from nerve terminals occurs through exocytosis of presynaptic vesicles, a process that is triggered by depolarization leading to entry of Ca2+. Release, triggered by depolarization and entry of Ca2+, allows dopamine to act on postsynaptic dopamine receptors (DAR).

Metabolism

Once dopamine is in the synaptic cleft, its actions may be terminated by reuptake through a membrane carrier protein, a process antagonized by drugs such as cocaine. Alternatively, dopamine can be degraded by the sequential actions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) to yield two metabolic products, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA). In human beings, HVA is the primary product of the metabolism of dopamine

Dopamine receptors

Dopamine receptors have key roles in many processes, including the control of motivation, learning, and fine motor movement, as well as modulation of neuroendocrine signaling. Abnormal dopamine receptor signaling and dopaminergic nerve function is implicated in several neuropsychiatric disorders. Thus, dopamine receptors are common neurologic drug targets.

Antipsychotics- dopamine receptor antagonists while psychostimulants- indirect agonists of dopamine receptors. At the cellular level, the actions of dopamine depend on receptor subtype expression.

The dopamine receptors share several structural features, including the presence of seven alpha-helical segments capable of spanning the cell membrane. This structure identifies the dopamine receptors as members of the larger superfamily of seven-transmembrane-region receptor proteins. All members of this superfamily act through guanine nucleotide-binding proteins.

The actions of dopamine in the brain are mediated by a family of dopamine receptor proteins. Two types of dopamine receptors were identified in the mammalian brain using pharmacological techniques:

D1 receptors family, which stimulate the synthesis of the intracellular second messenger cyclic AMP, and

D2 receptors family, which inhibit cyclic AMP synthesis.

The five dopamine receptors can be divided into two groups on the basis of their pharmacological and structural properties (Fig. 2).

SNpc, substantia nigra pars compacta; cAMP-cyclic AMP; psi-voltage.

Fig. 2 Distribution and characteristics of dopamine receptors.

D1-like family (excitatory)

The D1 and D5 proteins have a long intracellular carboxy-terminal tail and are members of the pharmacologically defined D1 class. Activation of the D1-like family receptors is coupled to the G protein Gαs, which subsequently activates adenylyl cyclase, increasing the intracellular concentration of the second messenger Cyclic adenosine monophosphate (cAMP). Increased cAMP in neurons is typically excitatory and can induce an action potential by modulating the activity of ion channels.

D2-like family (inhibitory)

The D2, D3, and D4 receptors share a large third intracellular loop and are of the D2 class. D2-like activation is coupled to the G protein Gαi, which subsequently increased phosphodiesterase activity. Phosphodiesterases break down cAMP, producing an inhibitory effect in neurons. They decrease cyclic AMP formation and modulate K+ and Ca2+ currents.

D1-like receptor agonists

Fenoldopam, Piribedil, Ibopamine, SKF 3893, Apomorphine

Therapeutic uses of D1-like receptor agonists

* Decreases peripheral resistance

* Inducing lowering of arterial blood pressure-increases in heart rate and increases in sympathetic tone

* Increases in activity of the rennin-aldosterone system

D2-like receptor agonists

Bromocriptine, Pergolid, Lisuride, Guinpirole, Carmoxirole

Therapeutic uses of D2-like receptor agonists

* Used for treating Parkinson’s disease

* Inhibits prolactin release (which decreases tumor size)

D1-like receptor antagonists

SCH23390, Clozapine (used for treating schizophrenia)

D2-like receptor antagonists

Metoclopramid, Domperidone (Gastric Motility Disorders), Sulpiride, Haloperidol,

Dopamine receptors in disease

Dysfunction of dopaminergic neurotransmission in the CNS has been implicated in a variety of neuropsychiatric disorders, including Tourette's syndrome (inherited neurological disorders- tic disorder (involuntary movement), Parkinson's disease, schizophrenia, Attention-deficit hyperactivity disorder (ADHD), and drug and alcohol dependence.

Attention-deficit hyperactivity disorder

Dopamine receptors have been recognized as important components in the etiology of ADHD for many years. Drugs used to treat ADHD, including methylphenidate and amphetamine, have significant effects on dopamine signaling in the brain. Studies of gene association have implicated several genes within dopamine signaling pathways; in particular, the D4.7 variant of D4 has been consistently shown to be more frequent in ADHD patients. The D4.7 allele has suppressed gene expression compared to other variants.

Drug abuse

Dopamine is the primary neurotransmitter involved in the reward pathways in the brain. Thus, drugs that increase dopamine signaling may produce euphoric effects. Cocaine and methamphetamine—two examples of such drugs—alter the functionality of the dopamine transporter (DAT), the protein responsible for removing dopamine from the neural synapse. When DAT activity is blocked, the synapse floods with dopamine and increases dopaminergic signaling. When this occurs, particularly in the nucleus accumbens, increased D1 and D2 receptor signaling mediates the "rewarding" stimulus of drug intake.

Schizophrenia

While there is evidence that the dopamine system is involved in schizophrenia, the theory that hyperactive dopaminergic signal transduction induces the disease is controversial. Psychostimulants, such as amphetamine and cocaine, induce dramatic changes in dopamine signaling; large doses and prolonged usage can induce symptoms that resemble schizophrenia. Additionally, many antipsychotic drugs target dopamine receptors, especially D2 receptors.

Genetic hypertension

Dopamine receptor mutations can cause genetic hypertension in humans. This can occur in animal models and humans with defective dopamine receptor activity, particularly D1.

Gamma amino butyric acid (GABA) and its receptor

GABA

GABA is the major inhibitory amino acid transmitter of the mammalian central nervous system and it is present in some 40% of all neurones.

Synthesis, storage and function

Gamma amino butyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. It is found in almost every region of brain, and is formed through the activity of the enzyme glutamic acid decarboxylase (GAD).

GAD catalyzes the formation of GABA from glutamic acid. The synthesis of GABA is linked to the Kreb's cycle. GAD requires vitamin B6 (pyridoxal phosphate) as a cofactor, which can be used to regulate the levels of GABA. GABA is destroyed by a transamination reaction, in which the amino group is transferred to alpha-oxoglutaric acid (to yield glutamate), with the production of succinic semialdehyde, and then succinic acid. The reaction is catalysed by GABA transaminase.

Vigabatrine, a GABA agonist, is used to treat epilepsy by inhibiting GABA transaminase.

GABA-ergic neurons and astrocytes take up GABA by specific transporters, and it is this, rather than GABA transaminase, that removes the GABA after it has been released. GABA transport is inhibited by Guvacine and Nipecotic acid.

Compounds such as the competitive GAD inhibitor allylglycine, inhibit GABA formation and cause convulsions due to the lack of GABA activity.

Sodium valproate (or valproic acid) on the other hand, blocks GABA transaminase activity, thereby elevating GABA levels, and thus alleviating seizures. Sodium valproate is useful in the treatment of epilepsy and bipolar mood disorders. Another strategy is to block GABA-transaminase with g-acetylenic GABA, thereby increasing the concentration of GABA at the synapse.

GABA receptors

Structure and Pharmacology

The GABA receptors are a class of receptors that respond to the neurotransmitter gamma aminobutyric acid (GABA), the chief inhibitory neurotransmitter in the vertebrate central nervous system. There are three classes of GABA receptors: GABAA, GABAB, and GABAС.

GABAA and GABAС receptors are ligand-gated ion channels (also known as ionotropic receptors), whereas GABAB receptors are G protein-coupled receptors (also known as metabotropic receptors).

Ligand-gated ion channels

GABAA

It has long been recognized that the fast response of neurons to GABA that is blocked by bicuculline and picrotoxin is due to direct activation of an anion channel.

This channel was subsequently termed the GABAA receptor. Fast-responding GABA receptors are members of family of Cys-loop ligand-gated ion channels. Members of this superfamily, which includes nicotinic acetylcholine receptors, GABAA and GABAС receptors, glycine and 5-HT3 receptors, possess a characteristic loop formed by a disulphide bond between two cysteine residues.

The GABAA receptor consists of three separate subunits with an alpha2, beta2, gamma arrangement similar to that found for neuronal nicotinic receptors. The ligand binding site is located at the interface between the alpha and beta subunits. The benzodiazepine binding site is located at a similar level at the interface between the alpha and gamma subunits. Each subunit is comprised of four hydrophobic sequences which span the membrane, with a large extracellular amino terminus containing the binding site and a carboxyl terminus located intracellularly.

Electrophysiological studies demonstrated that the activation of the receptor resulted in increased chloride conductance of the cell membrane with the concentration-response curve exhibiting positive cooperativity, consistent with the presence of at least two agonist binding sites on the receptor molecule. The agonist induced current decreased on continued exposure to high agonist concentrations suggesting that these receptors undergo desensitisation.

GABAC

In addition to the GABAA receptors there is a distinct class of ligand gated ion channels that are activated by GABA; referred to as the GABAC receptor. The natural agonist GABA is about an order of magnitude more potent at the GABA receptors than at the most common of the GABA receptors. The GABA receptors are activated by cis-aminocrotonic acid (CACA), which is not recognised by either the GABAA or GABAC receptors, suggesting that they recognise the partially folded conformation of GABA.

GABAC receptors are not blocked by bicuculline and do not recognise the benzodiazepines, barbiturates or the neuroactive steroids but, like GABAA receptors are blocked by picrotoxin, while 1,2,5,6-tetrahydropyridine-4-yl methyl phosphinic acid appears to inhibit GABAC receptors selectively.

However, molecular cloning studies have revealed that this pharmacological profile is remarkably similar to that exhibited by the rho-subunits when expressed ectopically. Two homologous subunits, rho1 and rho2, have been identified in man and these can be expressed as homomers or heteromers, but do not co-assemble with any of the GABA receptor subunits.

Common characteristics

In ionotropic GABAA and GABAС receptors, binding of GABA molecules to their binding sites in the extracellular part of receptor triggers opening of a chloride ion-selective pore.

Opening of a chloride conductance drives the membrane potential towards the reversal potential of the Cl¯ ion which is about –80 mV in neurons, inhibiting the firing of new action potentials.

However, there are numerous reports on GABAA receptors, which are actually excitatory. This phenomenon is due to increased intracellular concentration of Cl¯ ions either during development of the nervous system or in certain cell populations.

After this period of development, a Chloride pump is up-regulated and inserted into the cell membrane, pumping Cl- ions into the extracellular space of the cell. Further openings via GABA binding to the receptor then produce inhibitory responses. Over-excitation of this receptor induces receptor remodeling and the eventual invagination of the GABA receptor. As a result, further GABA binding becomes inhibited and IPSPs are no longer relevant.

G protein coupled receptor: GABAB

A slow response to GABA is mediated by GABAB receptors, originally defined on the basis of pharmacological properties.

In studies focused on the control of neurotransmitter release, it was noted that a GABA receptor was responsible for modulating evoked release in a variety of isolated tissue preparations. This ability of GABA to inhibit neurotransmitter release from these preparations was not blocked by bicuculline, was not mimicked by isoguvacine, and was not dependent on Cl¯, all of which are characteristic of the GABAA receptor. The most striking discovery was the finding that baclofen (β-parachlorophenyl GABA), a clinically employed spasmolytic mimicked, in a stereoselective manner, the effect of GABA.

Later ligand-binding studies provided direct evidence of binding sites for baclofen on central neuronal membranes. cDNA cloning confirmed that the GABAB receptor belongs to the family of G-protein coupled receptors. Additional information on GABAB receptors has been reviewed elsewhere.

Drugs acting on GABA receptors

Below is the table showing the effector pathway, agonists and antagonists of GABA receptors.

GABAA

GABA binds to GABAA receptors in the extended form as demonstrated by the activity of trans 4-aminocrotonoic acid and the lower activity of cis 4-aminocrotonoic acid.

Muscimol is a naturally occurring compound isolated from Amanita muscaria and acts as a GABAA agonist. The isoxazole ring also is found in the GABAA agonist tetrahydroisoxazolopyridinol (THIP).

Bicucullin acts as a direct antagonist at the GABAA receptor as does SR 42641.

GABAB

Baclofen is direct agonist at GABAB receptors, which are coupled to G proteins. GABAB receptors may regulate Ca2+ and K+ influx through the Gi/o family of G proteins and act presynaptically to inhibit the release of excitatory amino acids such as glutamate. Baclofen is orally active as a muscle relaxant and has been used in the treatment of rigidity and spasticity of cerebral palsy. Phaclofen is a weak partial agonist, while saclofen is an antagonist at GABAB receptors.

Anxiolytics: Benzodiazepines

Benzodiazepines represent a class of compounds collectively referred to as anxiolytics. Benzodiazepines modulate the binding of GABA to the GABAA receptor. Benzodiazepines increase the binding of GABA to GABAA receptors and promote Cl- influx.

All the overt effects of the benzodiazepines: sedative, anxiolytic, anticonvulsant, muscle relaxant and amnesic, are produced via the GABAA receptors.

Diazepam (Valium) is one of the most widely prescribed sedatives on the market.

Benzodiazepines exhibit anxiolytic activity due to their ability to promote GABA binding. They act as indirect agonists. Several ligands can block the actions of benzodiazepines, including flumazenil, a benzodiazepine antagonist.

Anticonvulsant drugs

Anticonvulsants are used to treat the various forms of epilepsy, which is characterized by excessive neuronal firing in the cortical and temporal lobe regions of the brain. Electroencephalograms (EEG) can pick up the rhythmic discharge of neurons from electrodes placed on the scalp. Rhythmic discharges of spikes and slow waves characterize the EEG during seizures.

The benzodiazepines have an anticonvulsant action in addition to the anxiolytic activity. Recent work has helped distinguish between the two roles for the benzodiazepines. Picrotoxin is a convulsant which interacts with the GABA receptor complex and blocks the Cl- ionophore.

Diphenylhydantoin (phenytoin) is useful in the treatment of epilepsy. Phenytoin stabilizes the neuron against the excitation associated with seizure activity, without producing sedation.

Beta-adrenoceptors

রাজশাহী বিশ্ববিদ্যালয়ের ফার্মেসী বিভাগের এম. ফার্ম সিলেবাস অনুযায়ী প্রণীতঃ
Syllabus- Molecular and cellular mechanisms of 1) Glutamate receptors, 2) GABA and its receptors, 3) Catecholamine receptors ( alpha- and beta-adrenoceptors, dopamine receptors), 4) Acetylcholine receptors (nicotinic and muscarinic receptors), 5) Opioid receptors.

β-Adrenoceptor Subtypes

The β -adrenoceptors were initially divided into β1 and β2-adrenoceptors defined in terms of agonist potencies, β1-adrenoceptors demonstrated equal affinity for adrenaline and nor- adrenaline while β2-adrenoceptors displayed a higher selectivity for nor-adrenaline than for adrenaline. The discovery of these receptor subtypes led to the development of selective agonists and antagonists for each subtype.

The story does not end there; further experimentation using β-antagonists exposed another receptor subtype which appeared to be insensitive to typical β-adrenoceptor antagonists this was classified as β3-adrenoceptor. More recent pharmacological evidence is now emerging in support of a further receptor subtype β4-adrenoceptor, although as yet there are no selective compounds for this particular subtype.

Characteristics of β-adrenoceptors

β1 adrenoceptors

All β1 adrenoceptors mediate responses to noradrenaline released from the sympathetic nerve terminals and to circulating adrenaline. β1-adrenoceptors were originally defined based on their rank order of potency of these endogenous agonists and by the synthetic agonist isoprenaline. Isoprenaline has shown to be the most potent of the three agonists with adrenaline and noradrenaline showing equal affinity for this particular receptor.

Subsequently, synthetic antagonists have been identified for β1 receptors these include the non-selective propranolol as well as the more selective compounds like metoprolol, practolol, atenolol and betaxolol. The selective β1-adrenoceptor agonists so far identified (denopamine, xamoterol and Ro 363) have limited utility as pharmacological tools because of low selectivity and/or efficacy.

β1-Adrenoceptor Location and Function

β1-adrenoceptors are largely postsynaptic and are located mainly in the heart but are also found in platelets, the salivary glands and the non-sphincter part of the gastrointestinal tract (GIT). They can however be found presynaptically. Activation causes an increase in the rate and contractile force of the heart, relaxation of the non-sphincter part of the GIT, aggregation of platelets and amylase secretion from the salivary glands. Presynaptically, their activation causes an increase in noradrenaline release.

The major ligands used for the autoradiographic localisation of β-adrenoceptor subtypes are [125I] cyanopindolol, [3H] dihydroalprenolol and [125I] pindolol. Of these [125I] cyanopindolol is the most commonly used because of its high affinity, ease of preparation, relatively short exposure time, and selectivity for β-adrenoceptors.

Catecholamines acting on β1 adrenoceptors mediate a number of tissue responses including increases in cardiac rate and force of contraction, stimulation of renin secretion, relaxation of coronary arteries and relaxation of gastrointestinal smooth muscle.

Transduction Mechanisms

All known adrenoceptors are coupled to their effector systems by guanine nucleotide binding proteins (G proteins). β1 adrenoceptors are positively coupled to the membrane bound enzyme adenylate cyclase via activation of Gs G-protein. Activation of adenylate cyclase produces alterations in cellular activity by increasing intracellular levels of cAMP. In the myocardial cell increases in cAMP cause more L-type voltage sensitive Ca2+ channels to open in response to depolarisation. Calcium then enters the cell by this route with each action potential and causes an immediate rise in the concentration of intracellular calcium.

Raised levels of intracellular calcium stimulate further calcium release from the sarcoplasmic reticulum (see diagram above). Activation of the contractile machinery is due partly to this influx of calcium but mainly to the secondary release from the sarcoplasmic reticulum.

Both the heart rate (chronotropic effect) and the force of contraction (inotropic effect) are increased, resulting in a markedly increased cardiac output and cardiac oxygen consumption. The cardiac efficiency is reduced. Catecholamines can also cause disturbance of the cardiac rhythm, cumulating in ventricular fibrillation.

β2 adrenoceptors

β2 adrenoceptors are mainly associated with causing relaxation of smooth muscle in a variety of tissues (see diagram below). The β2 subtype has a high affinity for the endogenous agonist adrenaline. Synthetic β2 agonists include terbutaline, salbutamol, salmeterol and zinterol, all of which have proved therapeutically useful in the treatment of asthma. Selective β2 antagonist have also been identified these include butoxamine and ICI118 551.

Diagram adapted from Pharmacology (4th edition) Rang Dale and Ritter

β2-Adrenoceptor Location and Function

β2-receptors are also mainly postsynaptic and are located on a number of tissues including blood vessels, bronchi, GIT, skeletal muscle, liver and mast cell. Activation results in vasodilatation, bronchodilation, relaxation of the GIT, glycogenolysis in the liver, tremor in skeletal muscle and inhibition of histamine release from mast cells.

The location of β2 adrenoceptors can be identified using the radiolabelled ligand [3H] dihydroalprenolol or [125I] iodopindolol and its analogues.

β2 adrenoceptors have been cloned and expressed in a variety of species. Cloning was first achieved by screening hamster genomic libraries with oligonucleotides complimentary to peptide fragments of purified hamster lung β2 adrenoceptors. This yielded a clone that when expressed had functional and radioligand-binding characteristics consistent with those of the β2 adrenoceptors. Furthermore there appears to be only minor species differences between these clones, with approximately 87 to 93% overall amino acid identity.

Catecholamines acting on β2 adrenoceptors mediate a number of tissue responses. Bronchial smooth muscle is strongly dilated by activation of β2 receptors. Uterine smooth muscle responds in a similar way and β2 agonist are frequently used to delay premature labour. Skeletal muscle is also affected by adrenaline acting on β2 receptors though the effect is far less dramatic than that on the heart. The twitch tension of fast contracting fibres (white muscle) is increased by adrenaline, particularly if the muscle is fatigued, whereas the twitch of slow (red) muscle is reduced.

In man adrenaline and other β2 agonists cause a marked tremor, this is demonstrated in the shakiness that accompanies fear and excitement or the excessive use of β2 agonists (treatment of asthma being an example of this). β2 agonists also cause long term changes in expression of the sarcoplasmic reticulum proteins that control contraction kinetics and thereby increase the rate and force of contraction of skeletal muscle. Clenbuterol is a β2 agonist, which acts in this way and has been used illegally by "sportsmen" to enhance performance.

Histamine release is also inhibited by catecholamines acting apparently at β2 receptors on mast cells and the release of glucose from the liver is also controlled by β2 receptors leading to glycogenolysis.

Transduction Mechanisms

β2 adrenoceptors are also positively coupled to the membrane bound enzyme adenylate cyclase via activation of Gs G-protein. β2 adrenoceptor activation causes relaxation of smooth muscle through the activation of adenylate cyclase. Stimulation of adenylate cyclase produces alterations in cellular activity by increasing levels of cAMP. The resulting increased levels of cAMP activate protein kinase A, which phosphorylates and inactivates myocin light chain kinase (MLCK), the contractile machinery of smooth muscle.

β3 adrenoceptors

The β3 adrenoceptor has a profile quite distinct from that of β1 and β2 adrenergic receptors. Rodents, humans and other mammals share many of the characteristic β3 properties, although observable species-species differences have been identified. The naturally occurring variant of the human β3 receptor was correlated with hereditary obesity in Pima Indians, in Japanese individuals and in Western obese patients. Weight gain was also observed in female mice whose β3 gene had been disrupted.

These examples provided a picture of the important role of the β3 receptor in the regulation of lipid metabolism and as a possible target for drugs to treat certain forms of obesity.

β3-Adrenoceptor Location and Function

β3-adreceptors are expressed predominately in adipose tissue. Activation is proposed to be involved with noradrenaline induced changes in energy metabolism via lipolysis and thermogenesis.

Early antagonist/agonist studies of β adrenergic receptors suggested the existence of atypical responses clearly different from effects mediated by the β1 and β2 adrenoceptors. The new agonist BR37344 stimulated lipolysis in rodent brown adipocytes even in the presence of the antagonist propranolol. This and other discoveries led to the notion that additional receptors existed in fat, in the gut, in the heart and possibly in skeletal muscle. Cloning, sequencing and expression of the β3 gene in various species has now served to confirm the existence of this receptor.

The β3 adrenoceptor is composed of a single 408 amino acid residue peptide chain that belongs to the family of G-protein coupled receptors. The G-protein receptors are characterised by seven hydrophobic stretches of about 22 to 28 residues forming seven transmembrane segments. The transmembrane (TM) regions are linked with three intracellular and three extracellular loops. The amino acid (N) terminal of these receptors is located extracellularly and is glycosylated. The carboxy (C) terminal is intracellular and the case of the β3 receptors does not have any phosphorylation sites.

Transduction Mechanisms

For all β-adrenoceptors transduction is via G-proteins coupled to the intracellular second messenger adenylate cyclase. All β-receptors are positively coupled to adenylate cyclase via activation of Gs G-protein. However, activation of the β2 and β3-adrenoceptors results in stimulation or stimulation and inhibition of adenylate cyclase. Activation of the β1 and β4 receptor results in an increase in the formation of cAMP and the subsequent stimulation of cAMP-dependent protein kinase.

Comparing β3 with β3 receptors of other species revealed a high degree of sequence homology approximately 80-90% between human, bovine, rodent and canine. The human, monkey and bovine β3 receptors are closer to each other than any of the rodents sequences, in particular in transmembrane segment one.

Molecular Biology of β-adrenoceptors

Extensive studies on the molecular features of the β-adrenoceptors have been carried out and the β-adrenoceptor is now among the best defined of the G-protein-linked superfamily of receptors.

The human β-adrenoceptors (β1, β2 and β3) are derived from at least three distinct genes and comprise of 477, 413 and 408 amino acids respectively. All three β-adrenoceptors show high amino acid sequence homology for each receptor between species. For example the β1 receptor of the mouse and rat are 90% homologous to the human β1. Similarly the mouse β2 receptor is 95% homologous to the human β2. However there are marked sequence differences between the subtypes. The β1 shows 48.9% homology with β2 and 50.7% homology with β3, whereas β2 show 45.5% homology with the β3.

The G-protein superfamilies are characterised by their seven hydrophobic transmembrane spanning domains. Three extracellular and three cytoplasmic (intracellular) loops, with a glycosylated extracellular amine (N) terminus and cytoplasmic carboxy (C) terminus link the transmembrane domains. The seven transmembrane domains arrange in the membrane to form a "pocket".

A great deal of the research done to determine the functional domains of the β-adrenoceptors has been carried out on the β2 receptor. Many of these studies have involved the preparation and expression of site directed mutations (deletion or substitution) of particular amino acids and also the synthesis of chimeric receptors.

β1

Three amino acids-Leu110, Thr117 and Val120 in TM2 of the β1-AR were identified as major determinants for β1-AR selective agonists. TM4 is responsible for β1-selective binding of noradrenaline.

β2

Important residues involved in ligand binding also include Asp79, 113, and 130. Asp79 appears to be essential for the binding of high affinity agonist but is not required for the binding of antagonists. Asp113 located in the third transmembrane domain is vital for binding of both agonist and antagonist in the β2 receptor. Substitution of this residue with a Glu results in a marked decrease in the ability of the receptor to couple to adenylate cyclase. Substitution of Asp130 increases the affinity of the receptor for the agonist but reduces the ability of the receptor to couple to the G-protein.

Other important amino acids associated with binding include Ser203, 204 & 207 in TM5; Tyr326 and Phe289, 290 alteration of these residues by substitution resulted in an increased affinity for agonist with no effect on antagonist binding. Ser204 forms hydrogen bonds with the meta hydroxy group of catecholamines; similarly Ser207 interacts with the para hydroxy group group of catecholamines.

Chimeric receptor studies revealed that transmembrane domains six and seven were important in determining the specificity of antagonist binding. Asn293 in TM6 interacts with the β-OH group of β-AR ligands and is responsible for stereoselectivity.

Residues of the third intracellular loop are involved in the coupling of the receptor to the Gs-protein, where the deletion of amino acids 222-229 and 239-272 caused complete loss of coupling to adenylate cyclase.

β3

Computer modelling has defined an image of the β3 ligand-binding site. At least four of the seven transmembrane domains are essential for ligand binding (see diagram below). The amino acids that are involved were identified by site-directed mutagenesis and photoaffinty labelling these are Asp117 in TM3, a residue found to be essential for binding all biogenic amines. Ser169 in TM4, is thought to form a hydrogen bonds with the hydroxyl of the ethanolamine side-chain. Ser209 and Ser212 in TM5, also located in many biogenic amine receptors, are thought to form hydrogen bonds with the hydroxyls of the catechol side chain. Also Phe309 in TM6, involved in hydrophobic interactions with the aromatic ring of catecholamines. Two of the three TM domains are involved in Gs activation, TM2, which contain Asp83, and TM7, which contains Tyr336.

Attempts to explain why several β2 antagonists behave as β3 agonist have not yet been successful. β3-adrenergic receptors appear to contain less bulky residues, and could therefore accommodate the larger β1/β2 antagonists. Mutagenesis has been used to substitute the small Gly residue with the larger Phe residue at position 53 in the β3 receptor; this change however was not enough to convert the β3 agonist to an antagonist.

The Gs interaction site on β3 is situated in the intracellular region, mainly the membrane proximal regions of the second and third (i2, i3) intracellular loops and the carboxy-terminal domains. Several reports propose that the β3 receptor may be coupled to more than one second messenger system. It has been shown that β3 receptors in rat adipocytes interact with both Gs and Gi proteins. The β3 adrenergic receptor in the septum of the human heart has also been reported to link with the Gi protein, resulting in a negative inotropic effect. This is in contrast to previous observations that always associated β3-adrenoceptors with positive inotropism in the heart.

All of the β-adrenoceptors have a glycosylated extracellular site on the N terminus; these sites are not associated with binding or signal transduction however it is thought that they are important for insertion of the receptor into the membrane. β adrenoceptors also contain a number of Cys residues (Cys106, 184, 190 & 191) which are thought to form disulphide bonds between the hydrophilic extracellular loops, which stabilise the receptor conformation.

β4-adrenergic receptor

β4-receptors are localised in cardiac tissue with activation causing an increase in heart rate and force.

Clinical Uses

Adrenergic drugs are used in the treatment of a wide range of medical conditions. Including the use of β2-receptor selective agonists in the treatment of asthma and other related bronchospastic conditions examples of these drugs include salbutamol and salmeterol. Beta-blocker drugs are commonly used in the treatment of angina pectoris, cardiac arrhythmia and for the long-term treatment of patients who survive myocardial infarction. β-receptor antagonists have also been used as anti-hypertensive for a number of years. Beta -blockers have also proven useful in the treatment of conditions such as migraine, anxiety disorders, hyperthyroidism, alcohol withdrawal and when applied topically are useful in the treatment of glaucoma and ocular hypertension.