G-Proteins and GPCRs G-proteins are “molecular clocks” that initiate or participate in many pathways GPCRs (G-protein coupled receptors) are 7TM (7-helix transmembrane) receptors that act through … Heterotrimeric G-protein transducers localized at the membrane that control… Many downstream effectors GPCRs are common pharmaceutical targets Vision is the prototype, rhodopsin the structural model Sources: Helmreich, Gomperts, Voet and Voet Science’s STKE Jason Kahn: G proteins and GPCRs

Importance in Pharmacology Klabunde and Hessler, ChemBioChem 2002, 3, 928 - 944 Jason Kahn: G proteins and GPCRs

Pharma II One often sees the claim that 50% (or 30%, or 60%, or 70%) of all drugs (or the top 100 prescription drugs, or total drug sales) target GPCRs. As far as I can tell this claim originated in the early-mid 1990’s and has just been propagated, may no longer be true. The preceding page adds up to only about $25,000,000,000, a small fraction of GDP. But a huge variety of disorders are treated by inhibiting GPCRs. Remarkably, there is only one structure of a GPCR: inhibitors have been developed by tried and true screening and other big-pharma methods. We should understand something about how GPCRs and G-proteins work! Jason Kahn: G proteins and GPCRs

Fundamental Idea of G-protein Signalling V+V 3/e: Figure 19-13. Activation/deactivation cycle for hormonally stimulated AC. (There are also G proteins that inhibit adenylate cyclase upon G-protein activation.) Jason Kahn: G proteins and GPCRs

GTP-binding proteins and G-proteins G-proteins and GTP-binding proteins are slow GTPases (2-3 min-1). When GDP is bound, they are inactive, when GTP is bound, they are active. Therefore the G proteins have built-in clocks: the stimulus lasts for as long as the GTP does, and then the protein reverts to an inactive form. A molecular egg timer. G-proteins typically act through binding to other proteins. They act as transducers between the receptors and the downstream effectors like adenylate cyclase. “G-protein” refers to the heterotrimeric partners of the GPCRs, GTP-binding protein refers to monomeric proteins like the Ras proto-oncogene product, Arf, Rho, EF-Tu. The heterotrimeric G-proteins are anchored to the membrane by palmitoyl or myristoyl fatty acid chains. G-proteins and GTP binding proteins are activated by guanine nucleotide exchange factors (GEFs). The 7TM GPCR is the GEF for the heterotrimeric G-proteins. Exchange typically activates because the cellular [GTP] is about 10x [GDP]. Jason Kahn: G proteins and GPCRs

Heterotrimeric G-protein Structure The , , and  subunits are about 45, 37, and 9 kD respectively. The G subunit binds GTP and GDP. Activation via nucleotide exchange weakens or dissociates the G subunit from the G, which remain tightly associated with each other. Structures of GDP-bound and GTPS-bound forms have been solved. Part of G resembles Ras proteins, the remainder interacts with G  is a 7-bladed WD40-domain propeller bg Gi•GDP a V+V 3/e GDP Viewed from the top (from the membrane) Jason Kahn: G proteins and GPCRs

Conformational Changes with GTP vs. GDP Switch regions mediate  interactions with . GTP binding weakens the interaction of  and , hence binding of  stabilizes GDP binding Switch regions and other loops mediate effector binding. Jason Kahn: G proteins and GPCRs

GTP/GDP cycle The GDP-bound heterotrimer is the resting state. It is often bound to an inactive effector. Association with the activated receptor (the GEF) is transient: The GTP-bound form of  is released. Hence the receptor can activate several G-proteins, conferring amplification. There might be soluble GEFs as well. Upon exchange the effector is active. The GTPase activity is also increased by RGS (regulator or G-protein signalling) proteins, which are analogous to the GAP (GTPase activating proteins) seen in Ras signalling. The GTPase activity of the  subunit is accelerated by contact with the effector, ensuring that the signal turns off rapidly. This is probably due to allosteric activation of the GAP activity of the RGS proteins in a ternary complex. GDI proteins (guanine nucleotide dissociation inhibitors) can prolong signalling if they bind the GTP-bound form. Kinetic regulation: on/off = active/inactive = [G•GTP]/[G•GDP] = k(GDP release) / k(GTP hydrolysis) The G-proteins also influence the receptor: since the GTP-bound form is released, the binding of agonists (activating ligands) is weakened by GTPS while the binding of inverse agonists (ligands that stabilize the inactive receptor) is enhanced. Jason Kahn: G proteins and GPCRs

Expanded GTP/GDP Cycle See A. M. Preininger, H. E. Hamm, G protein signaling: Insights from new structures. Sci. STKE 2004, re3 (2004). Jason Kahn: G proteins and GPCRs

Diversity of Responses There are hundreds to thousands of GPCRs in humans, many of which are olfactory receptors The different possible effects of G-protein activation are mediated by different , , and  subunits. The 16 or so G subunits fall into four main phylogenetic classes: GS (including olfactory receptors olf) activates adenylate cyclase Gi (including visual transducin Gt = t) inactivates adenylate cyclase or activates cGMP phosphodiesterase Gq/11 activates phospholipase C  (PLC) and also the Btk tyrosine kinase G12 can activate or inactivate RhoGEF Obviously a great deal of crosstalk among pathways is possible. There are 5  subtypes and at least 12  subtypes, leading to a large number of possible  combinations. Besides maintaining the •GDP in an inactive state (acting as a GDI to prevent GDP dissociation), the  pair can act to activate K+ channels independently of the  subunit. Furthermore, the  subunit can target the ARK G-protein coupled receptor kinase to its cognate GPCR, leading to specific down-regulation of receptor signalling. Classic negative feedback: homologous desensitization. Receptors can also be phosphorylated by PKA (protein kinase A, which we recall is activated by cAMP), hence leading to general down-regulation of the GS class: negative feedback called heterologous desensitization. Jason Kahn: G proteins and GPCRs

Diversity of G-protein Expression No more than about 10 Gs are produced in any one cell, leading to tissue specificity in the signalling pathways. From a ~50 page “20 questions” feature in Nature Reviews in Drug Discovery, 3 (2004), pp. 577-624 Jason Kahn: G proteins and GPCRs

The -adrenergic receptor was the second 7TM cloned Most ligands interact with more than one receptor isoform From Iyengar, STKE From Philpp M and Hein L (2004) Pharmacol Ther 101: 65-74 The -adrenergic receptor was the second 7TM cloned Table 1: Phenotypes of Mice deficient in adrenergic receptor subtypes

Heterotrimeric G protein Pathways From Iyengar, STKE Coupling to different receptor isoforms lead to different G protein pathways and different biological effects

What about the receptors? They respond to a huge variety of extracellular agonists, antagonists, and inverse agonists. Some have never before been made, e.g. NCEs that have a smell. The process of “deorphaning” is very slow due to lack of structures. Image shows families organized according to G-protein target, ligand, and phylogeny. Jason Kahn: G proteins and GPCRs

Natural Ligands Jason Kahn: G proteins and GPCRs

Effect of Ligand Binding Include hormones, scents, neurotransmitters, etc. Agonists activate receptors. Antagonists block the action of agonists but do not reduce the basal receptor activity. Inverse agonists decrease the activity of the receptor. If there is a cryptic agonist present, it can be difficult to distinguish an antagonist from an inverse agonist. Jason Kahn: G proteins and GPCRs

Re-expanded GDP/GTP Cycle Agonist binding G-protein binding Activation Jason Kahn: G proteins and GPCRs

Receptors Have 7 Membrane-Spanning -Helices Gomperts V+V 3/e Jason Kahn: G proteins and GPCRs

Space filling model of Ga interacting with Gbg From Iyengar, STKE Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.

A structural cartoon of G protein interaction with receptor Hamm J Biol Chem. 1998 Jan 9;273(2):669-672. From Iyengar, STKE