monomeric gpcr as a functional unit marc chabre and marc le maire presented by : naveena sivaram

Monomeric GPCR as a Functional UnitMarc Chabre and Marc le Maire

Presented by : Naveena Sivaram

Overview

• Challenges monomer vs dimer concept

• Study found to strengthen & support - monomeric model of rhodopsin and other class 1a GPCR’s

• Proposed a molecular model for functional coupling of Rhodopsin monomeric model with G-Protein Heterotrimer

(A) GPCRs have a central common core made of seven transmembrane helices (TM-I to -VII) connected by three intracellular (i1, i2, i3) and three extracellular (e1, e2, e3) loops. The diversity of messages which activate those receptors is an illustration of their evolutionary success. (B) Illustration of the central core of rhodopsin. The core is The core is viewed from the cytoplasmviewed from the cytoplasm. The length and orientation of the TMs are deduced from the two-dimensional crystal of bovine and frog rhodopsin (Unger et al., 1997). The N- and C-terminal of i2 (including the DRY sequence) and i3 are included in TM-III and -VI. The core is represented under its 'active conformation'. The TM-VI and -VII lean out of the structure, the TM-VI turn by 30% on its axis (clockwise as viewed from the cytoplasm) (Bourne, 1997). This opens a cleft in the central core in which G proteins can find their way. i2 and i3 loops are the two main loops engaged in G protein recognition and activation.

EMBO J. 18: 1723-1729 (1999)

GPCR encoded for >1000 genes, represent app.1% of human gemone

Family 1 contains most GPCRs including receptors for odorants, small ligands, peptide hormone and glycoprotein. Disulphide bridge connects e1 and e2 and palmitoylated cysteine in C-terminal.

Family 2 GPCRs have relative long N-terminal that contains several cysteines (network of disulphide bridge). Examples include glucagon, GnRH and PTH receptors.

Family 3 GPCRs have very large span N-terminal sequences and C-terminal tail. The ligand binding domain is located in the N-treminus. The i3 loop is short and highly conserved. Representative samples are mGluR, Ca2+-sensing and GABA-B receptor.

GTP binding protein (G-protein)

• Definition: binding the guanine nucleotides (GTP and GDP) and process intrinsic GTPase activity

• physiological meaning: essential role in signal transduction, vesicle transport, cytoskeletal assembly and cell growth

• classification: heterotrimer G protein () and small G proteins (ras, raf, rho or rab protein)

Consequence of receptor activation

a. Alteration in GPCR TM domain: TM3 and TM6 spin and conformation change

b. G-protein dissociation ( separate from ) c. signal transfer to cytoplasmic effectors ( adenylyl

cyclase, PLC etc. )d. Activation of effectorse. signal generation downwards

Early Studies

• Hydrodynamic studies with Triton X100 by Osborne etal

• Small angle neutron expt.. in polyoxyethylene glycol

• Late biochemist Hermann Kuhn – functional interaction between R* & Gt

• Though one exception for monomeric model was stated in Nature review article in 1981.

Recent Studies

• Medina etal.. – oligomeric state using cross-linking expt’s – Dm solubilized rhodopsin was dimeric

• Jastrzebska etal – cross linking expt’s

• Park & Wells – M2 muscranic cholinergic receptor

• Mesnier and Baneres – cross linking expt’s

Evidences

• None of crystallographic studies – suggest existence of a dimer

• Structure is of an inactive rhodopsin

• Inconsistencies in the structure of Gt

Molecular Model of Interaction RhodopsinMonomer and a Gt

• Park etal argues that Gt is larger than rhodopsin monomer

• Gt and Gt C-terminal peptides doesn’t interact with inactive rhodopsin & interacts with photo activated rhodopsin at two different sites.

• Proposed Model by Marc etal.

Figure : Models for a rhodopsin monomer in the lipid membrane, a transducin heterotrimer attached to the lipid membrane, and a complex between a photoactivated rhodopsin and a transducin.

Future Studies

• Crystallization of a complex with R* and nucleotide free Gt

• Obtaining a high resolution structure