2. What is G-protein??
• Also known as guanine nucleotide-binding proteins.
• Family of protein that act as a molecular switches inside the cell.
• Activity regulated by factors that controls their ability to bind to and
hydrolyze GTP into GDP.
• When they are bound to GTP, they are 'on', and, when they are bound
to GDP, they are 'off'. G proteins belong to the larger group of enzymes
called GTPase.
• There are two classes of G proteins:
a. monomeric small GTPase
b. heterotrimeric G protein complexes (alpha (α), beta (β) and gamma
(γ) subunit)
3. G-Protein Coupled Receptor
• 7 trans membrane helices connected by alternating cytosolic and extra
cellular loop
• C terminal: inside the cell. N terminal : extra cellular region
• Extra cellular portion has unique messenger binding site. Cytosolic loop
allow receptor to interact with G protein.
• The eventual effect of agonist -induced activation is a change in the
relative orientations of the TM helices (likened to a twisting motion)
leading to a wider intracellular surface and "revelation" of residues of
the intracellular helices and TM domains crucial to signal transduction
function (i.e., G-protein coupling).
• Inverse agonists and antagonists may also bind to a number of different
sites, but the eventual effect must be prevention of this TM helix
reorientation
4.
5. Genetic variations may be due to
1.Sequence variations of the human genome
• Introduces variability in genetic make-up
• Suspected to play a main role in diseases & variable response in
drug therapy
• Polymorphism- refers to sequence variation leading to occurrence of
two or more clearly different forms.
• Single nucleotide polymorphism accounts for approx. 80% of all
sequence variations.
6. 2. Structure & function of GPCRs
• Comprises a large class of membrane proteins – encoded by approx.
600 human genes.
• Molecular architecture might permit the prediction of functionally
relevant domains where sequence variations are more likely to alter
receptor function.
• Normally, TM domains are highly conserved, the loops are variable in
sequence & length, &the C- and N-terminals tails represents the most
diverse elements.
7. 3. GPCR coupling to G proteins and other signaling
pathways
• GPCR thought to be couple to heterotrimeric G - proteins composed of α,
β and γ subunits.
• It display considerable heterogenecity, with a predicted number of 27
different α, 5 β and 13 γ subunits.
• Main sites of contact between receptor and G proteins include the third
intracellular loop, but i1, i2 and the C- terminus have also been reported to
contribute G protein coupling
• Proteins like protein kinases, arrestin & phosphatases modulates
receptor functions at distinct domains that are possible targets for
polymorphic effects.
8. 4. GPCR binding pockets
• Ca++, acetyl choline, glutamate, bradykinin, prostaglandins & the large
polypeptide FSH bind to the same site.
• Distinct binding sites appear to exist, either embedded within the
pocket formed by the 7- TMD bundle within the membrane, at pockets
formed by the extracellular loops, or in the N- terminus.
• The thrombin receptor family represents a special case whereas the
protease activity of the ligand thrombin cleaves a portion of the N-
terminus.
• The newly generated N-terminus then serves as a tethered ligand.
• GPCRs appear to be activated by ligand binding to many different sites
of the protein.
9. • At the opioid receptors, peptide endorphins bind primarily to the
extracellular loops, whereas opioid alkaloids dock deep into the 7-TMD
core.
• Sequence variation in the receptor protein can affect ligand binding or the
strctural integrity of the receptor , indirectly changing ligand binding .
Human μ opioid receptor
10. 5. Spontaneous GPCR signalling
• Exchange of single amino acid residues can lead to constitutive receptor
activation.
• Considerable number of human polymorphisms enhance signalling
(gain of function) or even activate the receptor constitutively, causing
serious genetic disorders.
11. 6. Multiple receptor conformations with distinct
functions
• GPCRs are flexible structures and may accommodate ligands in
various ways.
• It exists in multiple conformations.
• Discrete signalling pathways are triggered by discrete conformational
states of GPCR
13. Impaired or enhanced agonist signalling efficacy
• Several inactivating sequence variants of peptide receptors have been
associated with congenital disorders.
• For example,
A point mutation causing truncation of thyrotropin
stimulating hormone receptor leads to leydig’s cell
hyperplasia.(activating mutation) Truncated TM5, D578G,
T398M
Inactivating mutations of the ACTH receptor are associated
with familial glucocorticoid deficiency . The mutation occurs in
the large N-terminus , the binding site for glycoprotein
hormone receptor, leading to toxic multinodular goiter. S120R,
R201Stop, S74I, V254C
14. V2 vasopressin recptors
• A number of mutations in the gene encoding the V2 vasopressin receptor
leads to functionally inactive receptor protein and are causative for
nephrogenic diabetes insipidus.(missense mutations)
• This a clear indication that receptor activity depends on intact
signalling pathways. (multiple SNPs; decreased ligand binding;
R113W; R137H)
15.
16. Thromboxane A2 Receptor
• This receptor performs an essential role in haemostasis by inducing
platelet aggregation.
• An R60L amino acid substitution in the first cytoplasmic loop of TBXA2
receptor causes a dominantly inherited bleeding disorder characterised
by defective platelet response to TBXA2.
• This leads to decreased agonist-induced second messenger formation.
17.
18. P2Y 12ADP Receptor
• This receptor sub-type is shown to be the target for anti-thrombotic drugs
such as ticlodipine & clopidogrel.
• 2-nucleotide deletion in a region mapping to the end of TMD6, associated
with a rare bleeding disorder.
19. Chemokine reeptors
• Fusin and CKR5 have been identified as a co- receptors for the cellular
entry of HIV. Similarly , certain chemokines were found to block HIV
entry into cells.
• Natural resistance can be either by high endogenous levels of
chemokines or by mutations of the receptors.
• A 32 bp deletion in CKR5 leading to a frame shift and a non functional
protein appeared to protect homozygous carriers against HIV infection &
blocking its entry.
• Val 64 substitution with Ile was shown to result in heterodimerisation of
CCR2 with CCR5 or CXCR4, thereby promoting resistance to AIDS.
20. Biogenic amine receptors
• The R16G substitution in the β2 adrenoreceptors has been associated with
nocturnal asthma whereas W64R in the β3 receptor expressed in
adipocytes are involved in energy metabolism – is linked with obesity.
22. Receptors Variant/Allele Disease/ Phenotype cellular
mechanism/ Event
Β1 adrenergic receptor R16G Nocturnal asthma;
Enhanced agonist promoted
down regulation of receptor
Β3 adrenergic
receptors
W64R Obesity
Luteinising hormone Truncated TM5 Leydig’s cell hyperplasia;
D578G Precocious puberty in male
children
FSH A189V Ovarian dysgenesis;
Altered protein folding;
inactivation of receptor
Thyrotropin (TSH) S120R, R201Stop,
S74I,
Glucocorticoid deficiency;
V254C altered/ loss of receptor
function/ reduced
expression
ACTH D727E Altered receptor
function/conformation;
Toxic multinodular goiter
Vasopressin V2 Multiple SNPs Nephrogenic diabetes insipidus;
23. Decreased ligand binding;
reduced expression of
receptor
Chemokine receptors CCR2
CCR3
CCR5
V64I
R275Q,L351P
CCR5P1 alleles
Delayed progression of AIDS
Unknown functional change
or influence on disease
Increased progression of
AIDS
Thromboxane A2 R60L Bleeding disorder
ADP receptor P2Y12 Del of 2 nt (TTCATT) in
coding region (end of
TMD6)
Bleeding disorder
