1. 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

2. Importance in Pharmacology
Klabunde and Hessler, ChemBioChem 2002, 3,
Jason Kahn: G proteins and GPCRs

3. 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

4. Fundamental Idea of G-protein Signalling
V+V 3/e: Figure 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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
Jason Kahn: G proteins and GPCRs

12. 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

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

14. 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

15. Natural Ligands
Jason Kahn: G proteins and GPCRs

16. 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

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

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

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

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