G PROTEIN COUPLED RECEPTOR (GPCR) G-protein-coupled receptors (GPCRs) • constitute a large and diverse family of proteins whose primary function is to transduce extracellular stimuli into intracellular signals • Among the largest and most diverse protein families in mammalian genomes. • On the basis of homology with rhodopsin, they are predicted to contain seven membrane-spanning helices, an extracellular N-terminus and an intracellular C-terminus. • This gives rise to their other names,: the 7-TM receptors or the heptahelical receptors. • GPCRs transduce extracellular stimuli to give intracellular signals through interaction of their intracellular domains with heterotrimeric G proteins. • This class of membrane proteins can respond to a wide range of agonists, including photon, amines, hormones, neurotransmitters and proteins. • Some agonists bind to the extracellular loops of the receptor, others may penetrate into the transmembrane region. GPCRs..contd • Their presence in the genomes of bacteria, yeast, plants, nematodes and other invertebrate groups argues in favor of a relatively early evolutionary origin of this group of molecules. • The diversity of GPCRs is dictated both by the multiplicity of stimuli (include light, neurotransmitters, odorants, biogenic amines, lipids, proteins, amino acids, hormones, nucleotides, chemokines) to which they respond and by the variety of intracellular signalling pathways they activate. • There are at least 18 different human G proteins to which GPCRs can be coupled. • These G proteins form heterotrimeric complexes with Gß subunits, of which there are at least 5 types, and G subunits, of which there are at least 11 types • • • • • Signal molecules A wide variety of ligands use GPCRs to stimulate cytoplasmic and nuclear targets through heterotrimeric G-protein-dependent and independent pathways. Biogenic amines: noradrenaline, dopamine, 5-HT, histamine, acetylcholine, epinephrine, norepinephrine Amino acids and ions: glutamate, Ca2+, GABA Lipids: LPA (lysophosphatidic acid), PAF (platelet-activating factor), prostaglandins, leukotrienes, anandamine, S1P (sphingosine-1phosphate) Peptides and proteins: Tripeptide N-formyl-Met-Leu-Phe, GnRH (gonadotropin-releasing hormone), angiotensin, bradykinin, thrombin, bombesin, glucagon, calcitonin, vasoactive intestinal peptides, PTH (parathyroid hormone), FSH (follicle-stimulating hormone), LH (leuteinizing hormone), TSH (thyroid-stimulating hormone), endorphins Nucleotides: adenosine nucleotides, adenine nucleotides, urdine nucleotides Others: light, odorants, pheromones, opiates, cannabinoids G-Protein-Coupled Receptor (GPCR): Structure and Function G-Protein-Coupled Receptor (GPCR) The largest family of integral membrane protein involved in many biological process and pathologies; 50% of all modern drugs and 25% of the top 200 best selling drugs are estimated to target GPCRs; Transduce the signals mediated by diverse signaling molecules, such as ions, peptides, lipids and photons, to induce different intracelluar function; Bind their ligand and to activate different G proteins; GPCR-Gα Fusion Protein Schematic of a G-protein-coupled receptor (GPCR)-Gα fusion protein STRUCTURE • The receptor's amino (N) terminal is extracellular (above the plane of the membrane), and its carboxyl (C) terminal intracellular. • The terminals are connected by a polypeptide chain that traverses the plane of the membrane seven times. • The hydrophobic transmembrane segments (light color) are designated by roman numerals (I–VII). • The agonist (Ag) approaches the receptor from the extracellular fluid and binds to a site surrounded by the transmembrane regions of the receptor protein. • G proteins (G) interact with cytoplasmic regions of the receptor, especially with portions of the third cytoplasmic loop between transmembrane regions V and VI. • The receptor's cytoplasmic terminal tail contains numerous serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated. • This phosphorylation may be associated with diminished receptor-G protein interaction. Transmembrane topology of a typical serpentine receptor . The receptor's amino (N) terminal is extracellular (above the plane of the membrane), and its carboxyl (C) terminal intracellular. The terminals are connected by a polypeptide chain that traverses the plane of the membrane seven times. The hydrophobic transmembrane segments (light color) are designated by roman numerals (I–VII). The agonist (Ag) approaches the receptor from the extracellular fluid and binds to a site surrounded by the transmembrane regions of the receptor protein. G proteins (G) interact with cytoplasmic regions of the receptor, especially with portions of the third cytoplasmic loop between transmembrane regions V and VI. The receptor's cytoplasmic terminal tail contains numerous serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated. This phosphorylation may be associated with diminished receptor-G protein interaction. G-protein cycling. Rate-limiting receptor-promoted GDP dissociation; GPCR CLASSIFICATION STRUCTURAL • • Three Families: A,B,C Based on sequence homology • Sequences within each family generally share over 25% sequence identity in the transmembrane core region, and a distinctive set of highly conserved residues and motifs. • Among the three families, little similarity is evident beyond the predicted 7TM architecture FUNCTIONAL • Six Classes : A,B,C,D,E,F • Based on chemical intercation • .According to the binding of GPCRs with different ligand types • The correlation between sub-family classification and the specific binding of GPCRs to their ligands can be computationally explored for Level 2 subfamily classification • • • • • GPCR CLASSIFICATION Class A: Rhodopsin like- Subfamilies Class B: Secretin like Class C: Metabotropic glutamate / pheromone Class D: Fungal pheromone Class E: cAMP receptors Functional classification GPCR Classes * Class A Rhodopsin like o Amine o Peptide o Hormone protein o (Rhod)opsin o Olfactory o Prostanoid o Nucleotide-like o Cannabinoid o Platelet activating factor o Gonadotropin-releasing hormone o Thyrotropin-releasing hormone & Secretagogue o Melatonin o Viral o Lysosphingolipid & LPA (EDG) o Leukotriene B4 receptor o Class A Orphan/other * Class B Secretin like o Calcitonin o Corticotropin releasing factor o Gastric inhibitory peptide o Glucagon o Growth hormone-releasing hormone o Parathyroid hormone o PACAP o Secretin o Vasoactive intestinal polypeptide o Diuretic hormone o EMR1 o Latrophilin o Brain-specific angiogenesis inhibitor (BAI) * Class C Metabotropic glutamate / pheromone o Metabotropic glutamate o Calcium-sensing like o Putative pheromone receptors o GABA-B o Orphan GPRC5 o Orphan GPCR6 o Bride of sevenless proteins (BOSS) o Taste receptors (T1R) * Class D Fungal pheromone o Fungal pheromone A-Factor like (STE2,STE3) o Fungal pheromone B like (BAR,BBR,RCB,PRA) o Fungal pheromone M- and P-Factor * Class E cAMP receptors * Frizzled/Smoothened family o frizzled o Smoothened Putative families: * Ocular albinism proteins * Insect odorant receptors * Plant Mlo receptors * Nematode chemoreceptors * Vomeronasal receptors (V1R & V3R) * Taste receptors T2R Orphans: Other families • Frizzled/Smoothened family Putative families: * Ocular albinism proteins * Insect odorant receptors * Plant Mlo receptors * Nematode chemoreceptors * Vomeronasal receptors (V1R & V3R) * Taste receptors T2R • Orphans: • * Putative / unclassified GPCRs • non-GPCR families: • * Class Z: Archaeal/bacterial/fungal opsins GPCR Ligands Rhodopsin family: amine receptors Acetylcholine (muscarinic) Adrenaline Dopamine Histamine Serotonin Octopamine Trace amine Rhodopsin family: other receptors Rhodopsin Olfactory Prostaglandin Prostacyclin Thromboxane Adenosine Purine / pyrimidine Cannabinoid Platelet activating factor Gonadotropin-releasing hormone Thyrotropin-releasing hormone • • • • • • • • • • • • • • • Rhodopsin family: peptide receptors • • Angiotensin • Apelin • Bombesin • Bradykinin • C5a anaphylatoxin • CC Chemokine • • CXC Chemokine • CX3C Chemokine • C Chemokine • Cholecystokinin • Endothelin • fMet-Leu-Phe • Galanin • • Ghrelin • • KiSS1-derived peptide Melanocortin Motilin Neuromedin U Neuropeptide FF Neuropeptide S Neuropeptide Y Neuropeptide W / neuropeptide B Neurotensin Orexigenic neuropeptide QRFP Opioid Orexin Oxytocin Prokineticin Somatostatin Tachykinin Urotensin II Vasopressin Protease-activated (thrombin) GPCR Ligands Melatonin Lysosphingolipid and LPA (EDG) Leukotriene B4 receptor SREB Mas proto-oncogene & Mas-related (MRGs) RDC1 EBV-induced Relaxin LGR like Free fatty acid G protein-coupled bile acid Nicotinic acid GPR GPR45 like Cysteinyl leukotriene Putative / unclassified Class A GPCRs Metabotropic glutamate family Glutamate (metabotropic) Extracellular calcium-sensing GABA-B Pheromone (V2R) Taste receptors (T1R) Orphan GPRC5 Orphan GPCR6 Bride of sevenless proteins (BOSS) Putative / unclassified Class C GPCRs Other families Frizzled / Smoothened family Ocular albinism proteins Vomeronasal receptors (V1R) Taste receptors (T2R) Insect odorant receptors Nematode chemoreceptors Plant Mlo receptors Fungal pheromone cAMP (Dictyostelium) Bacterial rhodopsin • • • • • • • • • • • • • • • • • Secretin family Calcitonin Corticotropin releasing factor Gastric inhibitory peptide Glucagon Growth hormone-releasing hormone Parathyroid hormone PACAP Secretin Vasoactive intestinal polypeptide EMR1 Latrophilin Brain-specific angiogenesis inhibitor (BAI) Methuselah-like proteins (MTH) Cadherin EGF LAG (CELSR) Putative / unclassified Class B GPCRs GPCR: Family A • The largest group • Includes the receptors for light (rhodopsin) and adrenaline (adrenergic receptors) and most other 7TM receptor types, including the olfactory subgroup. • The olfactory receptors constitute most of these sequences, but nearly 200 non-olfactory 7TM receptors that recognize over 80 distinct ligands have also been functionally characterized. GPCR: Family B • Contains only 25 members, including the receptors for the gastrointestinal peptide hormone family (secretin, glucagon, vasoactive intestinal peptide (VIP) and growth-hormone-releasing hormone), corticotropin-releasing hormone, calcitonin and parathyroid hormone • All family B receptors seen to couple mainly to activation of the effector adenylyl cyclase through the G protein Gs GPCR : Family C • Relatively small • Family C contains the metabotropic glutamate receptor family, the GABA receptor, and the calcium-sensing receptor, as well as some taste receptors. • All family C members have a very large extracellular amino terminus that seems to be crucial for ligand binding and activation. Three Families of GPCR Ligands: neurotranmitters (dopamine and serotonin) Ligands: hormones (glucagon, secretin, PTH) Example: mGluR, CaR • To date, the only member of the receptor superfamily for which a crystal structure has been solved is Rhodopsin. • This structure, which corresponds to the inactive receptor, confirms the presence of an anticlockwise bundle of 7 TM alpha helices (viewed from the intradiscal or extracellular side) that are connected by loops of varying lengths. General structure • N-terminal segment • Seven TMs, Three exoloops, Four cytoloops, • C-terminal segment. Family 1 GPCRs: Rhodopsin-like Receptors Represent the predominant class of GPCRs; Several highly conserved amino acid (red circles); Disulfide bridge between first and second extracellular loops (ECLs), palmitoylated cys in C-tail; The binding of small molecule ligands occures within the TM region; Example: Rhodopsin, Dopamine receptor, Chemokine receptors; • The rhodopsin crystal structure provides a structural basis for understanding the function of other G protein–coupled receptors (GPCRs). • The major structural motifs observed for rhodopsin are expected to carry over to other GPCRs, and the mechanism of transformation of the receptor from inactive to active forms is thus likely conserved. • Moreover, the high expression level of rhodopsin in the retina, its specific localization in the internal disks of the photoreceptor structures [termed rod outer segments (ROS)], and the lack of other highly abundant membrane proteins allow rhodopsin to be examined in the native disk membranes by a number of methods. • Evidence of the propensity of rhodopsin and, most likely, other GPCRs to dimerize, a property that may be pertinent to their function. Rhodopsin Rod cell specific visual pigment protein N 3 4 5 6 7 2 1 found in the vertebrate retina Responsible for achromatic vision under dim light conditions 348 amino acids, 7-TM, 11-cisretinaldehyde (chromophore, derived from Vit A) Posttransliational modifications: acetylation (M1), N-Glycosylation (N2, N15), S-palmitoylation (C322, C323) one ROS stack contains 1000-2000 discs • 6.4 million retinal cells (70% rods [B&W],