G PROTEIN COUPLED RECEPTORS

OVERVIEW

We are going to deal this topic, breaking them up into three smaller topics

GPCR

G-PROTEIN

SECOND MESSENGERS.

MUTATIONS IN GPCR AND G-PROTEINS.

G PROTEIN COUPLED RECEPTORS/ SERPENTINE RECEPTORS

G protein coupled receptors are also called as the seven trans membrane receptors.

STRUCTURE OF GPCR- Β ADRENERGIC RECEPTOR

GPCR

As the name suggests, This is a RECEPTOR coupled with G-Proteins.

Humans express over 800 GPCR and half of these are dedicated to sensory perception (smell, taste and vision).

LIGANDS THAT BIND TO THESE RECEPTORS

Neurotransmitters (eg.Ach) Biogenic amines (eg. NE) Ecosinoids and other lipid signaling molecule. Peptide hormones Opioids Amino acids (eg. GABA) Other miscellaneous peptide and protein

ligands.

RECEPTOR STRUCTURE

GPCRs are integral membrane proteins .

They possess seven membrane-spanning domains or transmembrane helices.

The receptors span the cell membrane 7 times.

These sense molecules outside the cell and activate signal transduction inside the cell.

DIMERIZATION OF GPCR

GPCRs undergo both homo- and heterodimerization and possibly oligomerization.

Heterodimerization can result in receptor units with altered pharmacology compared with either individual receptor.

Eg. opioid receptors can exist as homodimers of or receptors, or as heterodimers with distinctly different pharmacodynamic properties than either homodimer.

DIMERIZATION OF GPCR

Dimerization of receptors may regulate the affinity and specificity of the complex for G proteins, and regulate the sensitivity of the receptor to phosphorylation by receptor kinases and the binding of arrestin, events important in termination of the action of agonists and removal of receptors from the cell.

Dimerization also may permit binding of receptors to other regulatory proteins such as transcription factors.

Dimerization of single membrane spanning receptors is central to their activation.

WHY ARE THESE GPCR’S IMPORTANT IN PHARMACOLOGY?

Reasons -Because of their number, -Their wide array of ligands that bind to

them and -The variety of physiological functions they

mediate.

GPCR’s are the target of many drugs (Half of all non antibiotic prescription drugs act on these receptors).

FAMILIES OF GPCR:

3 Families

A – Rhodopsin family

B - Secretin/Glucagon receptor family eg. Peptide hormones.

C - Metabotropic Glutamate family eg. GABA , Glutamate.

RHODOPSIN RECEPTOR FAMILY

RLR are a family of proteins comprise of G protein-coupled receptors and are extremely sensitive to light.

It activates the G protein transducin (Gt) to activate the visual phototransduction pathway.

Mutation of the rhodopsin gene is a major contributor to various retinopathies.

SECRETIN RECEPTOR FAMILY 

The secretin-receptor family of GPCRs include Vasoactive intestinal peptide receptors and receptors for secretin, calcitonin and parathyroid hormone/parathyroid hormone-related peptides.

These receptors activate adenylyl cyclase and the phosphatidyl-inositol-calcium pathway.

METABOTROPIC GLUTAMATE FAMILY

The metabotropic glutamate receptors (mGluRs) are family C GPCR that participate in the modulation of synaptic transmission and neuronal excitability throughout the central nervous system.

They have been subdivided into three groups, based on intracellular signaling mechanisms.

Group I mGlu receptors (coupled to PLC and intracellular calcium signaling).

Group II Group III receptors are negatively coupled to adenylyl cyclase. These receptors are generally widely distributed throughout the mammalian brain with high levels in the cerebellum and thalamus.

7 TRANSMEMBRANE RECEPTOR

G PROTEINS

G- PROTEINS

GPCRs couple to a family of heterotrimeric GTP-binding regulatory proteins termed G proteins.

G proteins are signal transducers that convey the information that agonist is bound to the receptor from the receptor to one or more effector proteins.

G–protein-regulated effectors include enzymes such as adenylyl cyclase, phospholipase C, cyclic GMP phosphodiesterase (PDE6), and membrane ion channels selective for Ca2+ and K+ .

STRUCTURE OF G-PROTEINS

G Protein is a hetrotrimer.

The G protein heterotrimer is composed of

1. Guanine nucleotide-binding α subunit(which confers specific recognition to both receptors and effectors), and

2. An associated dimer of β and γ subunits that helps confer membrane localization of the G protein heterotrimer by prenylation of the subunit.

MECHANISM OF ACTION OF G PROTEINS

In the basal state of the receptor-heterotrimer complex, the α subunit contains bound GDP and the –α GDP: βγ complex is bound to the unliganded receptor

G PROTEIN- SUBUNITS

The G protein family is comprised of 23 α subunits (which are the products of 17

genes) 7 β subunits, and 12 γ subunits.

The α subunits fall into four families (Gs, Gi, Gq, and G12/13) which are responsible for coupling GPCRs to relatively distinct effectors.

ACTIVATION OF GPCR

When an agonist binds to a GPCR, there is a conformational change in the receptor that is transmitted from the ligand-binding pocket to the second and third intracellular loops of the receptor which couple to the G protein heterotrimer.

This conformational change causes the α subunit to exchange its bound GDP for GTP.

Binding of GTP activates the α subunit and causes it to release both the βγ dimer and the receptor, and active signaling molecules, both the GTP-bound α subunit and the βγ heterodimer become and active signaling molecules.

The interaction of the agonist-bound GPCR with the G protein is transient; following activation of one G protein, the receptor is freed to interact with other G proteins.

Depending on the nature of the subunit, the active, GTP-bound form binds to and regulates effectors such as adenylyl cyclase (via Gs ) or phospholipase C (via Gq ).

The subunit can regulate many effectors including ion channels and enzymes such as PI3-K. The G protein remains active until the GTP bound to the subunit is hydrolyzed to GDP.

The α subunit has a slow intrinsic rate of GTP hydrolysis that is modulated by a family of proteins termed regulators of G protein signaling (RGSs).

The RGS proteins greatly accelerate the hydrolysis of GTP and are potentially attractive drug targets.

Once the GDP bound to the α subunit is hydrolyzed to GDP, the βγ subunit and receptor recombine to form the inactive receptor-G protein heterotrimer basal complex that can be reactivated by another ligand-binding event.

TARGETS FOR G-PROTEINS

The main targets for G-proteins, through which GPCRs control different aspects of cell function.

adenylyl cyclase, the enzyme responsible for cAMP formation

phospholipase C, the enzyme responsible for inositol phosphate and diacylglycerol (DAG) formation

ion channels, particularly calcium and potassium channels

Rho A/Rho kinase, a system that controls the activity of many signalling pathways controlling cell growth and proliferation, smooth muscle contraction, etc.

SECOND MESSENGERS

SECOND MESSENGERS

Cyclic AMP PKA Cyclic AMP regulated Guanine Nucleotide

Exchange Factors. PKG PDES

CYCLIC AMP

Cyclic AMP is synthesized by adenylyl cyclase under the control of many GPCRs; stimulation is mediated by the Gs subunit, inhibition by the Gi subunit.

There are nine membrane-bound isoforms of adenylyl cyclase (AC) and one soluble isoform found in mammals.

Membrane-bound ACs exhibit basal enzymatic activity that is modulated by binding of GTP-liganded subunits of the stimulatory and inhibitory G proteins (Gs and Gi).

Numerous other regulatory interactions are possible, and these enzymes are catalogued based on their structural homology and their distinct regulation by G protein and subunits, Ca2+, protein kinases, and the actions of the diterpene forskolin.

Cyclic AMP generated by adenylyl cyclases has three major targets in most cells, the cyclic AMP dependent protein kinase (PKA), cAMP-regulated guanine nucleotide exchange factors termed EPACs (exchange factors directly activated by cAMP), and via PKA phosphorylation, a transcription factor termed CREB (cAMP response element binding protein

It is produced continuously and inactivated by hydrolysis to 5´-AMP, by the action of a family of enzymes known as phosphodiesterases (PDEs).

Many different drugs, hormones and neurotransmitters act on GPCRs and produce their effects by increasing or decreasing the catalytic activity of adenylyl cyclase, thus raising or lowering the concentration of cAMP within the cell.

Cyclic AMP regulates many aspects of cellular function including, for example, enzymes involved in energy metabolism, cell division and cell differentiation, ion transport, ion channels, and the contractile proteins in smooth muscle

PKA PKA can phosphorylate a diverse array of

physiological targets such as metabolic enzymes and transport proteins, and numerous regulatory proteins including other protein kinases, ion channels, and transcription factors.

G-PROTEIN MUTATIONS CAUSING DISEASE

MUTATIONS IN GPCR AND G PROTEINS

LOSS OF FUNCTION MUTATION.

Block the signaling by the corresponding agonist.

In endocrine signaling, loss-of-function mutations cause hormone resistance, mimicking hormone deficiency, whereas gain-of-function mutations mimic states of hormone excess. Defective G protein–mediated signaling can also lead to neoplasia and developmental and sensory abnormalities

DISEASES CAUSED BY GPCR LOSS-OF-FUNCTION MUTATIONS Cone opsins Color blindness X-linked; autosomal recessive Rhodopsin Retinitis pigmentosa Autosomal dominant; recessive V2 vasopressin Nephrogenic diabetes insipidus X-linked ACTH Familial ACTH resistance Autosomal recessive LH Male pseudohermaphroditism Autosomal recessive Ca2+ sensing Familial hypocalciuric hypercalcemia Autosomal

dominant Ca2+ sensing Neonatal hyperparathyroidism Autosomal recessive Endothelin-B Hirschsprung disease Complex FSH Hypergonadotropic ovarian failure Autosomal recessive TSH Congenital hypothyroidism Autosomal recessive TRH Central hypothyroidism Autosomal recessive GHRH Growth hormone deficiency Autosomal recessive GnRH Central hypogonadism Autosomal recessive Melanocortin 4 Extreme obesity Codominant PTH/PTHrP Blomstrand chondrodysplasia Autosomal recessive

CLASSIFICATION OF THE DISEASES ACCORDING THE MECHANISM

Inactive or absent Gs (α subunit)

Constitutively active Gs (α subunit)

Temperature-sensitive G αs

Constitutively active Gi α2

1.INACTIVE OR ABSENT GS (A SUBUNIT)

Pseudohypoparathyroidism-- type I (PHP-I), is an inherited human disease caused by mutational inactivation of the α subunit of Gs Multiple endocrine abnormalities in cAMP regulated organs

Occurs when bad gene inherited from mother

Pseudopseudohypoparathyroidism--clinically less severe syndrome, same as mutation

in one GsOccurs when bad gene inherited from

fatherBoth conditions--as protein levels about half of

normal

2. CONSTITUTIVELY ACTIVE GS (A SUBUNIT)

Tumors Mutations in αs --block GTPase activity,

cause constitutive activityas candidate oncogene (termed gsp)Activating mutations found in 40% of

growth hormone secreting pituitary adenomas; found in other endocrine tumors including pituitary, thyroid

McCune Albright SyndromeSomatic mutation in αs in early

embryonic development Patients mosaic for constitutively

active Gs (as)

MCCUNE-ALBRIGHT SYNDROME.

Somatic mutation of Gs alpha early in development

Effects of activating MSH and gonadotrophin receptors evident

3.TEMPERATURE-SENSITIVE ΑS--"TESTOTOXICOSIS"

Testotoxicosis ——the gain-of-function disorder

Symptoms: Males have general features of precocious puberty and hypoparathyroidism with a mutant as that is inactive at 37°C and constitutively active at testicular temperature

Both patients were found to contain a single amino acid substitution in one of the Gαisoforms. The alternation in amino acid sequence caused two effects on the mutant G protein.

precocious puberty--indicating premature testicular activation (normally testosterone production is stimulated by LH, a GPCR coupled to cAMP formation)

hypoparathyroidism--impaired responses to PTH, TSH causing PTH and thyroid abnormalities

At temperatures below normal body temperature, the mutant G protein remained in the active state, even in the absence of a bound ligand. In contrast, at normal body temperatures, the mutant G protein was inactive, both in the presence and absence of bound ligand, the testes, which are housed outside of the body’s core, have a lower temperature than the body’s visceral organs (33℃ versus 37 ℃).

PRECOCIOUS PUBERTY

Normally, the endocrine cells of the testes initiate testosterone production at the time of puberty in response to the pituitary hormone LH, which begins to be produced at that time.

The circulating LH binds to LH receptors on the surface of the testicular cells, inducing the synthesis of cAMP and subsequent production of the male sex hormone, the testicular cells of the patients bearing the G protein mutation were stimulated to synthesize cAMP in the absence of the LH ligand, leading to premature synthesis of testosterone and precocious puberty.

4. CONSTITUTIVELY ACTIVE GI Α2

TumorsMutations in ai2 --block GTPase

activity, cause constitutive activityαi2 candidate oncogene (termed gip)Activating mutations found in >30% of

adrenal, ovarian tumors

REFERENCE

Goodman and Gilman Textbook of Pharmacology.

Katzung Textbook of Pharmacology. Rang and Dale’s Textbook of Pharmacology. Internet sources.

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