Download - G protein coupled receptor

G Protein Coupled Receptor (GPCR) kinase

in health and disease

Dr.Mayank Agarwal [email protected]

CONTENT

1. Types of cell surface receptor2. Structure of G protein coupled receptor3. GCPRs activation mechanism4. G protein family classification5. Major type of G protein signaling pathways6. GPCR desensitization7. Classification of GPCR kinases8. Summary

Cell-surface receptors

ESSENTIAL CELL BIOLOGY 4ED, PG.538 FIG 16-17

ESSENTIAL CELL BIOLOGY 4ED, PG.538 FIG 16-17

GPCR: An Overview

• G proteins are intracellular signaling proteins that are named for their ability to bind to guanosine triphosphate (GTP)

• They also possess GTPase activity, the ability to hydrolyze GTP to GDP

• Two categories of G proteins are described: heterotrimeric G proteins and the homomeric G proteins

CELL AND MOLECULAR BIOLOGY : LIPPINCOTT ILLUSTRATED REVIEW SERIES 1ED, PG.160

All GPCRs have a similar structure


Alberts molecular biology of the cell 6ed, pg. 832

• The signal molecules that act on GPCRs are proteins, small peptides, derivatives of amino acids and fatty acids, photons of light and all the molecules that we can smell or taste.


Heteromeric G ProteinsNote that both the α and the γ subunits have covalently attached lipidmolecules (red tails) that help bind them tothe plasma membrane, and the α subunithas GDP bound.

α subunit contains the GTPase domain

alphahelical or “AH” domain, clamps the nucleotide in place

“Ras” domain provides one face of the nucleotide-binding pocket


Stimulation of GPCRs Activates G-Protein Subunits

In the unstimulated state, the receptor and the G protein are bothinactive. Although they are shown here as separate entities in the plasma membrane, in some cases at least, they are associated in a preformed complex.


Binding of an extracellular signal molecule to a GPCR changes the conformation of the receptor, which allows the receptor to bind and alter the conformation of a trimeric G protein. The AH domain of the G protein α subunit moves outward to open the nucleotide-binding site, thereby promoting dissociation of GDP


GTP binding then promotes closure of the nucleotide-binding site, triggering conformational changes that cause dissociation of the α subunit from the receptor and from the βγ complex


ESSENTIAL CELL BIOLOGY 4ED, PG.542

When a GPCR is activated, it acts like a guanine nucleotide exchange factor (GEF)

GTPase activity is greatly enhanced by the binding of the α subunit to a secondprotein, which can be either the target protein or a specific regulator of G protein signaling (RGS). RGS proteins act as α-subunit-specific GTPase-activating proteins (GAPs)


Cell physiology source book, 4ed, pg 88

Four Major Families of Trimeric G Proteins determined by amino acid sequence relatedness of the α subunits BRS cell biology histology 7ed, pg 9; Alberts molecular biology of the cell 6ed, pg. 846

Heterotrimeric G proteins signaling pathways

• Cyclic-AMP-Dependent Protein Kinase (PKA)

• Phospholipase C dependent IP3, DAG and calcium

• Direct regulation of ion channels

• cGMP Phosphodiesterase pathway

The activation of cAMP dependent protein kinase

Mammalian cells have at least two types of PKAs: type I is mainly in the cytosol, whereas type II is bound via its regulatory subunits and special anchoringproteins to the plasma membrane, nuclear membrane, mitochondrial outer membrane,and microtubules


Gs and Gi are targets for medically important bacterial toxins

• Cholera toxin, is an enzyme that causes ADP ribosylation of the α subunit so that it can no longer hydrolyze its bound GTP, causing it to remain in an active state that stimulates adenylyl cyclase indefinitely.

• The resulting prolonged elevation in cAMP concentration within intestinal epithelial cells causes a large efflux of Cl– and water into the gut, thereby causing the severe diarrhea that characterizes cholera.

• Pertussis toxin, catalyzes the ADP ribosylation of the α subunit of Gi, so that Gαi cannot inhibit adenylyl cyclase. Adenylyl cyclase remains active indefinitely, producing excess cAMPCELL AND MOLECULAR BIOLOGY : LIPPINCOTT ILLUSTRATED REVIEW SERIES 1ED, PG.163

The action of phospholipase C generates two small messenger molecules: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)


Calmodulin mediates many effects of intracellular calcium


Some G Proteins Directly Regulate Ion Channels

Essential cell biology 4ed, pg.543

cGMP phosphodiesterase pathway

Neuroscience exploring the brain 4ed, 315

Monomeric G proteins• Monomeric G proteins (=150 different ones) are important

intermediaries in numerous types of signal transduction systems and can be subdivided into five general families: Ras, Rho, Rab, Ran and Arf

• They are classified under enzyme coupled receptors

• Ras G proteins are homologous to the α subunits of heterotrimeric G proteins. They do not regulate membrane bound enzymes or induce the production of second messengers. Instead, their activation by GTP allows them to initiate a cytoplasmic phosphorylation cascade that terminates with activation of gene transcription.


GPCR Desensitization

• Three general modes of adaptation 1. receptor sequestration2. receptor down-regulation3. receptor inactivation

• In each case, the desensitization of the GPCRs depends on their phosphorylation by PKA, PKC, or a member of the family of GPCR kinases (GRKs)


The role of GPCR kinases and arrestins in GPCR desensitization

The GRKs phosphorylate multiple serines and threonines on a GPCR, but they do so only after ligand binding has activated the receptor, because it is the activated receptor that allosterically activates the GRK.


The binding of an arrestin to the phosphorylated receptor prevents the receptor from binding to its G protein


The β-arrestins recruit clathrin and the AP-2 complex, which target GPCRs for clathrin-mediated endocytosis

NATURE REVIEWS | NEUROSCIENCE VOLUME 2 | OCTOBER 2001 | 727-733

On the basis of sequence homology and tissue expression, GRKs are further separated into 3 subfamilies: rhodopsin kinases (GRKs 1 and 7); β adrenergic receptor kinases (GRKs 2 and 3); and the GRK4 subfamily (GRKs 4, 5, and 6)

G Protein Coupled Receptor Kinases as Therapeutic Targets in Cardiovascular Disease. Stephen L. Belmonte, Burns C. Blaxall

There are about 25 RGS proteins encoded in the human genome, each of which interacts with a particular set of G proteins

GPCR IN HEALTH AND DISEASES

CELL PHYSIOLOGY SOURCE BOOK 4ED PG 89

SUMMARY

MCQs

Q. Adenylyl cyclase is activated by a G protein. Which of the following second messengers will be generated?A. ATPB. cAMPC. CalciumD. DAGE. IP3

Correct answer = B.

cAMP is the second messenger generated by activated adenylyl cyclase that uses ATP as a substrate to produce the cAMP. Calcium, DAG, and IP3 are generated in response to phospholipase C activation. PIP2 is cleaved by phospholipase C to generate DAG and IP3.


Q. Manic-depressive illness may result from the overproduction of IP3 and DAG and the accompanying signaling processes in certain CNS cells. Lithium is often useful in treating this illness. Lithium most likely functions to inhibit

A. Adenylyl cyclase activity.B. Gαs protein function.C. Phospholipase C activity.D. PKA activity.E. Tyrosine kinase activity

Correct answer = C. Phospholipase C is the enzyme regulated by Gq that catalyzes the production of IP3 and DAG. Lithium’s inhibition of phospholipase C inhibits the production of IP3 and DAG. Adenylyl cyclase catalyzes the production of the second messenger cAMP when stimulated by active Gsα. PKA is regulated by cAMP. Tyrosine kinase activity is not involved in the production of DAG and IP3CELL AND MOLECULAR BIOLOGY :

LIPPINCOTT ILLUSTRATED REVIEW SERIES 1ED, PG.167

Q. A 6-month-old male patient presents with slight fever, rhinitis, and sneezing as well as forceful coughs ending with the loud inspiration (whoop). Bordetella pertussis was cultured from the nasopharynx. The toxin from this microorganism prevents the normal function of the Gαi protein in cells of the respiratory tract. Which of the following disruptions in cell signaling will result in the respiratory tract response to this infection?

A. Calcium being unable to bind to calmodulinB. Impaired IP3-stimulated release of calcium from endoplasmic reticulumC. Increased phospholipase C activity and PIP2 cleavageD. Increased stimulation of PKC activityE. Overproduction of cAMP from uninhibited adenylyl cyclase


Correct answer = E. Overproduction of cAMP from uninhibited adenylyl cyclase will occur when Gi is inhibited by pertussis toxin. Gi normally inhibits adenylyl cyclase. Calcium is released in response to activation of phospholipsase C. PKC is also activated as a consequence of phospholipase C activation.

Q. Protein kinase A

A. Activation by Ras stimulates gene transcription.B. Induces the release of calcium from endoplasmic reticulum.C. Is activated via Gq stimulation of phospholipase C.D. Phosphorylates protein substrates on serine/threonine residues.E. Stimulates the cleavage of PIP2


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