G-protein Receptors PDF

# Nature of Receptors Receptors are regulatory macromolecules, mostly proteins, though nucleic acids may also serve as receptors. Hundreds of receptor proteins have been isolated, purified, cloned and their primary amino acid (AA) sequence has been worked out. Molecular cloning has also helped in o...

# Nature of Receptors Receptors are regulatory macromolecules, mostly proteins, though nucleic acids may also serve as receptors. Hundreds of receptor proteins have been isolated, purified, cloned and their primary amino acid (AA) sequence has been worked out. Molecular cloning has also helped in obtaining the receptor protein in larger quantity to study its structure and properties, and in subclassifying receptors. The cell surface receptors with their coupling and effector proteins are considered to be floating in a sea of membrane lipids; the folding, orientation and topography of the system being determined by interactions between the lipophilic and hydrophilic domains of the peptide chains with solvent molecules (water on one side and lipids on the other). Nonpolar portions of the AA chain tend to bury within the membrane, while polar groups tend to come out in the aqueous medium. In such a delicately balanced system, it is not difficult to visualize that a small molecular ligand binding to one site in the receptor molecule could be capable of tripping the balance (by altering distribution of charges, etc.) and bringing about conformational changes at distant sites. Each of the five major families of receptors (described later) have a well defined common structural motif, while the individual receptors differ in the details of amino acid sequencing, length of intra/extracellular loops, etc. Majority of receptor molecules are made up of several non-identical subunits (heteropolymeric), and agonist binding has been shown to bring about changes in their quaternary structure or relative alignment of the subunits, e.g. on activation the subunits of nicotinic receptor move apart opening a centrally located cation channel. # General Pharmacology Many clinically useful drugs act upon physiological receptors which mediate responses to transmitters, hormones, autacoids and other endogenous signal molecules; examples are cholinergic, adrenergic, histaminergic, steroid, leukotriene, insulin and other such receptors. In addition, now some truly drug receptors have been described for which there are no known physiological ligands, e.g. benzodiazepine receptor, sulfonylurea receptor. Receptors for which no endogenous mediator or ligand is at present known are called 'Orphan receptors'. They, nevertheless, may prove to be targets for novel drugs yet to be developed. ## Receptor Subtypes The delineation of multiple types and subtypes of receptors for signal molecules has played an important role in the development of a number of targeted and more selective drugs. Even at an early stage of evolution of receptor pharmacology, it was observed that actions of acetylcholine could be grouped into 'muscarinic' and 'nicotinic' depending upon whether they were mimicked by the then known alkaloids muscarine or nicotine. Accordingly, they were said to be mediated by two types of cholinergic receptors, viz. muscarinic (M) or nicotinic (N); a concept strengthened by the finding that muscarinic actions were blocked by atropine, while nicotinic actions were blocked by curare. In a landmark study, Ahlquist (1948) divided adrenergic receptors into 'a' and 'B' on the basis of two distinct rankorder of potencies of adrenergic agonists. These receptors have now been further subdivided (M₁, M₂ ....Ms), (NM, N₂) (α₁, α₂) (β₁, β₂, β). Multiple subtypes of receptors for practically all transmitters, autacoids, hormones, etc. are now known and have paved the way for introduction of numerous clinically superior drugs. In many cases, receptor classification has provided sound explanation for differences observed in the actions of closely related drugs. The following criteria have been utilized in classifying receptors: - **Pharmacological criteria** Classification is based on relative potencies of selective agonists and antagonists. This is the classical and oldest approach with direct clinical bearing; was used in delineating M and N cholinergic, a and ẞ adrenergic, H₁ and H₂ histaminergic receptors, etc. - **Tissue distribution** The relative organ/tissue distribution is the basis for designating the subtype, e.g. the cardiac ẞ adrenergic receptors as ẞ₁, while bronchial as ẞ₂. This division was confirmed by selective agonists and antagonists as well as by molecular cloning. - **Ligand binding** Measurement of specific binding of high affinity radio-labelled ligand to cellular fragments (usually membranes) in vitro, and its displacement by various selective agonists/antagonists is used to delineate receptor subtypes. Multiple 5-HT receptors were distinguished by this approach. Autoradiography has helped in mapping distribution of receptor subtypes in the brain and other organs. - **Transducer pathway** Receptor subtypes may be distinguished by the mechanism through which their activation is linked to the response, e.g. M cholinergic receptor acts through G-proteins, while N cholinergic receptor gates influx of Na+ ions; a adrenergic receptor acts via IP-DAG pathway and by decreasing cAMP, while ẞ adrenergic receptor increases cAMP; GABA₁ receptor is a ligand gated Cl channel, while GABA₂ receptor increases K+ conductance through a G-protein. - **Molecular cloning** The receptor protein is cloned and its detailed amino acid sequence as well as three dimensional structure is worked out. Subtypes are designated on the basis of sequence homology. This approach has in the recent years resulted in a flood of receptor subtypes and several isoforms (which do not differ in ligand selectivity) of each subtype. The functional significance of many of these subtypes/isoforms is dubious. Even receptors without known ligands (orphan receptors) have been described. Application of so many approaches has thrown up several detailed, confusing and often conflicting classifications of receptors in the past. However, a consensus receptor classification is now decided on a continuing basis by an expert group of the International Union of Pharmacological Sciences (IUPHAR). # Mechanism of Drug Action; Receptor Pharmacology ## Silent Receptors These are sites which bind specific drugs but no pharmacological response is elicited. They are better called inert binding sites or sites of loss, e.g. plasma proteins which have binding sites for many drugs. To avoid confusion, the term receptor should be restricted to those regulatory binding sites which are capable of generating a response. ## Action-Effect Sequence 'Drug action' and 'drug effect' are often loosely used interchangeably, but are not synonymous. - **Drug action** It is the initial combination of the drug with its receptor resulting in a conformational change in the latter (in case of agonists), or prevention of conformational change through exclusion of the agonist (in case of antagonists). - **Drug effect** It is the ultimate change in biological function brought about as a consequence of drug action, through a series of intermediate steps (transducer). ## Receptors subserve two essential functions, viz, recognition of the specific ligand molecule and transduction of the signal into a response. Accordingly, the receptor molecule has a ligand binding domain (spatially and energetically suitable for binding the specific ligand) and an effector domain (Fig. 4.4) which undergoes a functional conformational change. These domains have now actually been identified in some receptors. The perturbation in the receptor molecule is variously translated into the response. The sequential relationship between drug action, transducer and drug effect can be seen in Fig. 4.1D and 4.6. ## Transducer Mechanisms Considerable progress has been made in the understanding of transducer mechanisms which in most instances have been found to be highly complex multistep processes that provide for amplification of the signal, as well as integration of concurrently received extra- and intra-cellular signals at each step. Because only a handful of transducer pathways are shared by a large number of receptors, the cell is able to generate an integrated response reflecting the sum total of diverse signal inputs. The transducer mechanisms can be grouped into 5 major categories. Receptors falling in one category also possess considerable structural homology, and belong to one super-family of receptors. ### 1. G-protein Coupled Receptors (GPCRs) These are a large family of cell membrane receptors which are linked to the effector (enzyme/channel/transporter) through one or more GTP-activated proteins (G-proteins) for response effectuation. All such receptors have a common pattern of structural organization (Fig. 4.5). The molecule has 7 a-helical membrane spanning hydrophobic amino acid (AA) segments which run into 3 extracellular and 3 intracellular loops. The agonist binding site is located somewhere between the helices on the extracellular face, while another recognition site formed by cytosolic segments binds the coupling G-protein. The G-proteins float in the membrane with their exposed domain lying in the cytosol, and are heterotrimeric in composition (α, ẞ and y subunits). In the inactive state GDP is bound to the α subunit at the exposed domain; activation through the receptor leads to displacement of GDP by GTP. The activated α-subunit carrying GTP dissociates from the other two subunits and either activates or inhibits the effector. The ẞy diamer has also been shown to activate receptor-operated K channels, to inhibit voltage gated Ca²+ channels and to promote GPCR desensitization at higher rates of activation. A number of G proteins distinguished by their α subunits have been described. The important ones with their action on the effector are: - Gs : Adenylyl cyclase activation, Ca²⁺ channel opening - Gi : Adenylyl cyclase inhibition, K⁺ channel opening - Go: Ca²⁺ channel inhibition - Gq: Phospholipase C activation A limited number of G-proteins are shared between different receptors and one receptor can utilize more than one G-protein (agonist pleotropy), e.g. the following couplers have been associated with different receptors. | Receptor | Coupler | |---|---| | Muscarinic M₁ | Gi, Go | | Muscarinic M₂, M₃ | Gq | | Dopamine D₂ | Gi, Go | | ẞ-adrenergic | Gs | | α₁-adrenergic | Gq | | α₂-adrenergic | Gi, Go | | GABAB | Gi, Go | | Serotonin 5-HT₁ | Gi, Go | | Serotonin 5-HT₂ | Gq | | Prostanoid | Gs, Gi, Gq | In addition, Gs is the coupler for histamine H₁, serotonin 5HT, glucagon, thyrotropin (TSH) and many other hormones, while Gi is utilized by opioid, cannabinoid and some other receptors. Moreover, a receptor can utilize different biochemical pathways in different tissues. The α-subunit has GTPase activity: the bound GTP is slowly hydrolysed to GDP: the α-subunit then dissociates from the effector to rejoin its other subunits, but not before the effector has been activated/inhibited for several seconds (much longer than the life-time of the activated receptor, which is in milliseconds) and the signal has been greatly amplified. The rate of GTP hydrolysis by the α subunit and thus the period for which it remains activated is regulated by another protein called 'regulator of G protein signaling' (RGS). The onset time of response through GPCRs is in seconds. ### There are three major effector pathways (Table 4.1) through which GPCRs function. #### (a) Adenylyl cyclase: cAMP pathway Activation of AC results in intracellular accumulation of second messenger cAMP (Fig. 4.6) which functions mainly through cAMP-dependent protein kinase (PK). The PK phosphorylates and alters the function of many enzymes, ion channels, transporters, transcription factors and structural proteins to manifest as increased contractility/impulse generation (heart), relaxation (smooth muscle), glycogenolysis, lipolysis, inhibition of secretion/mediator release, modulation of junctional transmission, water conservation by kidney, steroid hormone synthesis, etc. In addition, cAMP directly opens a specific type of membrane Ca²⁺ channel called cyclic nucleotide gated channel (CNG) in the heart, brain and kidney. The other mediators of cellular actions of cAMP are: cAMP response element binding protein (CREB) which is a transcription factor, cAMP regulated guanine nucleotide exchange factors called EPACs and certain transporters. Responses opposite to the above are produced when AC is inhibited through inhibitory Gi-protein. The action of cAMP is terminated intracellularly by phosphodiesterases (PDEs) which hydrolyse it to 5-AMP. Some isoforms of PDE (PDE, PDE) are selective for cAMP, while PDE, is selective for cGMP. #### Cyclic GMP (cGMP) as a second messenger In contrast to CAMP, the cGMP serves as an intracellular second messenger only in a limited number of tissues, such as vascular smooth muscle, intestinal mucosal cell and kidney. In these tissues it respectively mediates relaxation, inhibition of salt and water absorption as well as reduced proximal tubular Na+ reabsorption. There are two principal forms of guanylyl cyclases (GC) which generate cGMP, one cell membrane bound and the other cytosolic. However, none of these is regulated by a GPCR. The cell membrane bound GC is regulated by a transmembrane enzyme-linked receptor (described on p.58) for atrial natriuretic peptide (ANP). The cytosolic soluble GC in vascular smooth muscle is activated by nitric oxide (NO). After generation by the vascular endothelium NO diffuses into the adjacent smooth muscle cell and stimulates the soluble GC. Increased | | Adenylyl cyclase: CAMP activation | Adenylyl cyclase: CAMP inhibition | Phospholipase-IP/DAG activation | Channel regulation: Ca²+ opening | Channel regulation: Ca²+ closing | Channel regulation: K⁺ opening | |---|---|---|---|---|---|---| | Adrenergic-ẞ | Adrenergic-α₁, Histamine-H₂, Dopamine-D1, Glucagon, FSH & LH, ACTH, TSH | Adrenergic-α₂, Muscarinic-M₂, Dopamine-D2, 5-HT, GABAB, Opioid-μ, δ, Angiotensin-AT₁ | Adrenergic-α₁, Histamine-H₁, Muscarinic-M₁, M₃, 5-HT₂, Vasopressin-Oxytocin, Bradykinin-B₂, Angiotensin-AT₁, Prostaglandin-FP, EP₁, EP₃ | Adrenergic-β₁, Dopamine-D1 | Adrenergic-α₂, Dopamine-D2, Opioid-K, Somatostatin | Adrenergic-α₂, Dopamine-D2, 5-HT, GABA, Opioid-μ, δ, Adenosine-A₁ | | Prostaglandin-EP₂ | Prostaglandin-EP₁, Somatostatin, Adenosine-A₂ | Prostaglandin-EP₁, Thromboxane-TP, Leukotriene BLT, LT, Cholecystokinin-Gastrin, PAF | | | | | ### (b) Phospholipase C: IP-DAG pathway Activation of phospholipase Cβ (PLCβ) by the activated GTP carrying α subunit of Gq hydrolyses the membrane phospholipid phosphatidyl inositol 4,5-bisphosphate (PIP₂) to generate the second messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). The IP₃ being water soluble diffuses to the cytosol and mobilizes Ca²⁺ from endoplasmic reticular depots (Fig. 4.7). The lipophilic DAG remains within the membrane, but recruits protein kinase C (PKc) and activates it with the help of Ca²⁺. The activated PKc phosphorylates many intracellular proteins (depending on the type of effector cell) and mediates various physiological responses. So that it can serve signaling functions, the cytosolic concentration of Ca²⁺ is kept very low (~ 100 nM) by specific pumps located at the plasma membrane and at the endoplasmic reticulum. Triggered by IP₃, the released Ca²⁺ (third messenger in this setting) acts as a highly versatile regulator acting through calmodulin (CAM), PKc and other effectors-mediates/modulates smooth muscle contraction, glandular secretion/transmitter release, eicosanoid synthesis, neuronal excitability, intracellular movements, membrane function, metabolism, cell proliferation, etc. Signaling in this pathway is terminated by degradation of the second messengers. The IP₃ is dephosphorylated to inositol which is reutilized in the synthesis of PIP₂ (see Fig. 32.1), while DAG is partly converted back to phospholipids, and partly deacylated to arachidonic acid. Intracellular Ca²⁺ release has been found to occur in waves (Ca²⁺ mediated Ca²⁺ release from successive pools facilitated by inositol 1, 3, 4, 5-tetrakisphosphate-IP) and exhibits a variety of agonist and concentration dependent oscillatory patterns. The activation of different effectors may depend on the amplitude and pattern of these oscillations. Thus, the same intracellular messenger can trigger different responses depending on the nature and strength of the extracellular signal. ### (c) Channel regulation The activated G-proteins (Gs, Gi, Go) can also open or inhibit ionic channels specific for Ca²⁺ and K+, without the intervention of any second messenger like cAMP or IP₃, and bring about hyperpolarization/depolarization/changes in intracellular Ca²⁺ concentration. The Gs opens Ca²⁺ channels in myocardium and skeletal muscles, while Gi and Go open K⁺ channels in heart and smooth muscle as well as inhibit neuronal Ca²⁺ channels. Direct channel regulation is mostly the function of the ẞy dimer of the dissociated G protein. Physiological responses like changes in inotropy, chronotropy, transmitter release, neuronal activity and smooth muscle relaxation follow. Receptors found to regulate ionic channels through G-proteins are listed in Table 4.1.