GPCR Families

Question 1

GPCR Families and Representative Members

Answer 1

Family A - Rhodopsin
Family B - Secretin
Family C - Metabotropic Glutamate/Pheromone
Family D - Fungal Pheromone
Family E - cAMP receptors
Family F - Frizzled

Question 2

Basic GPCR structure

Answer 2

• N terminus extracellular
• 7 transmembrane helices
• C terminus intracellular

Question 3

Orthosteric vs Allosteric Binding Site

Answer 3

Orthosteric: Site where the endogenous agonist binds
Allosteric: Outside of the site where the endogenous agonist binds

Question 4

Family A (Rhodopsin) - General Overview

Answer 4

• largest GPCR family
• diverse range of ligands
  
  • biogenic amines
  • peptides
  • lipids
  • nucleotides
  • proteins
• small N terminus
• variety of orthosteric binding sites

Question 5

Family A (Rhodopsin) Classes

Answer 5

• alpha
  
  • opsin
  • aminergic
  • nucleoside
• beta
  
  • peptides/lipids
• gamma
  
  • peptides
• sigma
  
  • bigger proteins

Question 6

Family A GPCR’s extracellular domains

Answer 6

• large diversity in extracellular domains, especially in ECL2 (2nd extracellular loop)
• ligands can bind in a pocket that can be on the surface or as deep as ~14A into hydrophobic core of protein
• size, shape and amino acid composition of binding pockets explain ability of GPCR’s to distinguish between chemical signals
• differences in ECL2 lead to either an open binding pocket or one in which access is restricted to a small channel

Question 7

Family A - 2nd Extracellular Loop (ECL2)

Answer 7

• plays a role in the binding of orthosteric and allosteric ligands
• All ECL2 of Family A GPCR’s have a disulphide bond from ECL2 to top of transmembrane helix 3
• The beta-2 adrenergic receptor has a second intra-loop disulphide
• ECL2 structure varies across Family A
  
  • ECL2 of rhodopsin is made up of beta sheets and forms a lid over the binding pocket
  • ECL2 of beta-adrenoreceptor has a alpha-helix stabilised by a disulphide bond which lifts the loop away from the binding pocket

Question 8

Family A - Structural features of N terminus

Answer 8

• most receptor proteins within the rhodopsin family have short N termini without any common conserved domains
• protease activated receptors (PARs) have an intrinsic cleavage site at the N terminus
  
  • when cleaved by thrombin, reveals a tethered ligand that is able to activate the receptor
• leucine-rich repeat containing G-protein coupled receptors include the relaxin receptors and the glycoprotein hormone receptors

Question 9

Family A - structural features of the relaxin family peptide receptor RXFP1

Answer 9

RXFP1

• large extracellular domain, contains LDLa (low-density lipoprotein receptor class A domain) module connected to an LRR region (leucine-rich repeat) connected to the 7TM domain by a hinge region
• the primary high-affinity binding site for relaxin is located in the LRR region, but the peptide also interacts with a low-affinity binding site on the extracellular loops
• an intact LDLa module is essential for signalling

Question 10

Family B (Secretin) - General Overview

Answer 10

• Peptide ligands
• Larger N-terminus
• Orthosteric binding sites in N-terminus
• ~15 members of this family including:
  
  • amylin
  • calcitonin
  • calcitonin gene related peptide (CGRP)
  • Corticotropin releasing hormone (CRH)
  • Glucagon
  • Vasoactive intestinal peptide (VPAC)

Question 11

Family B - structural features

Answer 11

• antiparallel beta-sheet
• alpha-helix
• ligand

Question 12

Family B - Model of endogenous ligand binding and activation

Answer 12

Two step model of peptide ligand binding to family B GPCRs

1. C-terminal portion of ligand binds to N-terminal domain of receptor with high affinity and specificity
2. N-terminal of ligand binds to extracellular loop region of receptor
3. This activates the receptor and mediates intracellular interaction with G-proteins

Question 13

Family C (Metabotropic Glutamate) - General Overview

Answer 13

• Very large N-terminus (~560 amino acids)
  
  • Venus-fly trap domain
• 29 members of family include:
  
  • Metabotropic Glutamate R (1-8)
  • GABA R (GABAB1, GABAB2)
  • Calcium-Sensing R
  • Taste Receptors

Question 14

Family C - Structural Features of N-terminus

Answer 14

Venous Flytrap Domain (VFT)

• bi-lobular domain
• endogenous ligands bind in the hinge region between the 2 lobes
• shares structural similarity with bacterial periplasmic amino acid-binding proteins
• change conformation from open (inactive state) to closed (active state)

Cysteine-Rich Domain (CRD)

• links the venous flytrap domain to the seven transmembrane domain of the receptor
• a site of allosteric modulator binding

Seven Transmembrane Domain (7TM)

• a site of allosteric modulator binding
• couples to signalling pathways

Question 15

Family C - Function as Dimers

Answer 15

Homodimers

• Metabotropic Glutamate R (1-8)
• Calcium receptors

Heterodimers

• GABA B receptors (GABAB1, GABAB2)
• Taste Receptors
  
  • Sweet (Tas1R2, Tas1R3)
  • Umami (Tas1R1, Tas1R3)

Question 16

Family F (Frizzled) - General Overview

Answer 16

• large N-terminus
  
  • cysteine-rich domain
• orthosteric binding sites in cysteine-rich domain of N-terminus
• members of this family are involved in embryonic development and taste, include:
  
  • frizzled (1-10) (ligand WNT)
    beta-catenin signalling
  • smoothen (constitutively active)
  • bitter taste receptors

Question 17

Family F - Structural features of N-terminus

Answer 17

• smoothened receptor
• cysteine rich domain (CRD) - extracellular
• ligands bind to the CRD
• linker domain (LD)
• two N-linked glycans (NAG) - extracellular
• transmembrane domain
• inactivating point mutation V329F (in membrane)
• nine disulfide bridges