PCOL L4 GPCR Part 1

intracellular receptors

nuclear/steroid hormone receptors, soluble cellular enzymes, structural proteins, nucleic acids

intracellular transcription factors

lipophilic chemicals which diffuse across the plasma membrane and bind receptors in cytosol or in nucleus

activated receptors bind dna and start gene transcription

cell-surface receptors

ligand and voltage gated ion channels, GPCRs, receptor tyrosin kinases

how many subunits does nicotinic acetylcholine receptor have

5

what binds to to nicotinic acetylcholine receptor

ACh, a neurotransmitter, binds two alpha subunits of the nicotinic receptor

what happens after ACh binds to nicotinic acetylcholine receptor

channel opens and allows Na+ to pass

what are the three main parts of GPCR

transmembrane receptor, G protein, effector

what are the subunits of G proteins

3 subunits: alpha, beta, gamma

nucleotide mediators

extracellular nucleotides that act as signaling molecules (GTP and GDP)

act as molecular on/off switches for intracellular signaling

what is an enzymatic cytosolic domain

the intracellular portion of a transmembrane receptor that has catalytic (enzyme) activity

ie. tyrosine kinase, serine/threonine kinase, tyrosine phosphotase

what defines a transmembrane receptor

receptor that spans the cell membrane to cause signaling for intracellular responses

kinases

phosphorylation: add phosphate groups (PO4-) to proteins

act as the ON switch for signaling

What does tyrosine kinase do

phosphorylates tyrosine residues on target proteins

what is a receptor tyrosine kinase (RTK)

transmembrane receptor with intrinsic tyrosine kinase activity in its cytosolic domain

usually dimerize to activate the kinase for autophosphorylation (receptor phosphorylates itself after activation)

what residues are phosphorylated by serine/threonine kinases

serine and threonine residues

what is the role of phosphatases in signaling pathways

they remove phosphate groups to turn signaling OFF

what enzyme activity converts GTP to cGMP

guanylyl cyclase

which enzyme class is a major target in cancer therapy? what drug targets it?

tyrosine kinase, imatinib

signal transduction

process by which a cell converts information (signal) from one form to another

general steps of signal transduction

1. generation of signal (distant or local)

2. accumulation of signal at target site

3. binding of signal to effector protein (often a receptor)

4. induction of signaling event (conformation changes in effector, propogation of signal, can be fast or slow)

5. termination of signaling event

primary messengers

often extracellular ligands that bind and activate receptors (some intracellular receptors) and can have multiple functions

act on cells they were released from (autocrine), nearby cells (paracrine), distant cells (endocrine)

what are hormones and how do they reach their target organs

they are primary messengers released in small amounts from one site that travel thru bloodstream to act on distant target organs (endocrine signaling)

why must hormone receptors have high affinity?

they are diluted in circulation and bound to carrier proteins, resulting in low free concentrations requiring high-affinity receptors

growth factors

primary messengers that stimulate proliferation and also have high affinity receptors, tend to act locally and action can last days

cytokines

primary messengers that work on immune and other cell types to elicit biological responses

chemokines

cytokines that activate and recruit inflammatory cells by chemotaxis

vasoactive agents

primary messengers released at injury site to allow vasodilation and vascular permeability to recruit leukocytes/phagocytes

neurotransmitters

primary messengers released from presynaptic cells into the synaptic cleft that diffuse and bind receptors on presynaptic and postsynaptic cells

can be excitatory (glutamate) or inhibitory (gaba and glycine)

secondary messengers

intracellular molecules formed or activated in response to activation of receptors, often transmit signals from outside to inside of cell

ie. cAMP,GMP, Ca2+, DAG

signal amplification

binding of one ligand to receptor will activate multiple second messengers(adenyl cyclase → cAMP) which can activate multiple kinases which can then phosphorylate ode proteins leading to an amplified response

7 transmembrane receptors

single plypeptide chain traverses plasma membrane seven times (3 intracellular and 3 extracellular loops)

variety of ligands (growth factors, glucagon, epinephrine, prostaglandins, ACh, opioid peptides, etc

types of ligand-GPCR interactions

what are some processes and examples of GPCRs

vision, olfaction, cns, immune system, digestive system

ie. beta adrenergic, thyrotropin, glucagon and some types of dopamine and serotonin receptors

what are the 4 nucleotides and their functions

• CTP: phospholipid biosynthesis

• UTP: polysaccharide assembly

• ATP: metabolism & cell activity

• GTP: Receptor singling cofactor

what are g-proteins

gaunine nucleotide-binding receptors (also known as molecular switches or effectors)

they are the intermediary proteins in signal transduction

where are g-proteins found

along the inner surface of plasma membrane

when are g-proteins active/inactive

bound to gdp is inactive, bound to gtp is active

what is the difference between alpha, beta and gamma subunits of g-proteins

alpha subunit has the nucleotide binding site

beta and gamma act as 1 entity and are anchored to membrane by lipids

what are the g-protein activation steps

1. start as inactive heterotrimer: Gαβγ

2. agonist binding & receptor conformational change

3. recruit g protein to agonist-bound receptor

4. exchange gdp for gtp (on alpha subunit)

5. GTP-Gα and Gβγ subunits dissociate and activate different signaling cascades

what are the steps in g-protein inactivation

1. Gα subunit comes into contact with effector enzyme → GTPase activated

2. Gα subunit hydrollyze GTP to GDP

3. Gα and Gβγ reassociate

4. inactive form

why is the signal still amplified during g-protein inactivation

because hyrolysis of GTP is SLOW and allows stimulation of a number of effector molecules

what do the c-terminus and n-terminus on alpha subunit interact with

c-terminus interacts with receptors

n-terminus interacts with βγ subunit

after binding or gtp and dissociation of βγ dimer what happens to the α-subunit

surface of α-subunit is shownto effector molecule to interact

why is there so much diversity of heterotrimeric g proteins

because the α-subunit has different types

ie. universal expression (αs, αi, α11, αq)

ie. sensory (αt,αolf) differs from neurons (az)

what does the identity of the βγ dimer contribute to

coupling of G-proteins to particular receptors, localization, coupling and deactivation of a-subunit, reduce tendency of GDP to dissociate from a-subunit

how do βγ subunits act as signaling proteins

increase K+ channel activity, decrease Ca2+ channel activity

what enzyme catylyzes conversion of ATP to cAMP

adenylyl cyclase

what enzyme catalyzes the breakdown of cAMP to 5’-AMP (adenylate)

phosphodiesterase

how is adenylyl cyclase regulated

activated by certain Gα subtypes, forskolin, Ca2+-calmodulin by

deactivated by certain Gα subtypes

Gαs

stimulates production of cAMP from ATP by activating adenylyl cyclase

Gαi

inhibits production of cAMP from ATP by decreasing adenylyl cyclase

Gαq

increase phospholipase C activity

protein kinase function

turn enzymes on or off by phosphorylation

what exactly is added during phosphorylation

kinase add negatively charged phosphate groups (PO4-) to amino acids with an -OH group (hyrdoxyamino acids: serine, threonine, and sometimes tyrosine)

why do kinases specifically add to serines, threonines and tyrosines

because they are neutral, exposed on the protein surface and location at interfaces between protein subunits

protein kinase A (PKA)

soluble kinase with 2 catalytic and 2 regulatory domains (inactive tetramer form) that phosphorylates serine or threonine residues to produce multiple biological effects, has tissue-specific regulatory subunits,

activation of PKA

inactive tetramer binds to cAMP on regulatory subunits and detaches from active catalytic subunits

cAMP response element binding protein (CREB)

interacts with DNA at the cAMP response element (CRE) after being phosphorylated and activated by PKA

CRE

cAMP response element, 8 base pair palindromic sequence targeted by CREB

why is binding of cAMP onto PKA “cooperative”

because the activation energy of first cAMP is bind PKA is greatest, making the binding of each consecutive cAMP easier with a lower activation energy