Section 2 subsection 3 "Heterotrimeric G Proteins"

Heterotrimeric G protein part of

• superfamily of regulatory GTPases known as G proteins

G proteins are structurally similar

• Bind the guanine nucleotides GTP and GDP and hydrolyze GTP to GDP and Pi

Heterotrimeric G protein essential for

• signal transduction

• vesicle trafficking

• growth of actin microfilaments

• translation (ribosomal accessory factors)

• protein targeting (as components of signal recognition particles and SRP receptor)

Heterotrimeric G proteins in signal transduction with three major components

1) G proteins-couples receptors (GPCRs)

2) Heterotrimeric G proteins

3) Adenylate Cyclase (AC)

G protein-couples receptors (GPCRs)

• transmembrane proteins that bind their corresponding ligand

• on extracellular side

• induce conformation change on their cytoplasmic side

Heterotrimeric G protein location

• anchored on cytoplasmic side of the plasma membrane

Heterotrimeric G protein activated by

• A G protein Couples receptor (GPCR) when it binds its corresponding ligand

Adenylate Cyclase (AC)

• transmembrane enzyme

• transmembrane glycoproteins

Adenylate Cyclase in mammals

• 9 different isoforms or AC

AC isoforms are

• tissue specific

• have different regulatory properties

Adenylate Cyclase activated and sometimes inhibited by

• activated heterotrimeric G proteins

Activated Adenylate Cyclase

• catalyzes the synthesis of adenosine 3’, 5’-cyclic monophosphate (3’,5’ cyclic AMP or cAMP) from ATP

cAMP

• binds to a variety of proteins

• activate numerous cellular processes

• second messenger

The Adenylate cyclase Conserved Domains

• small N-terminal domain

• transmembrane domain (M1)

• 2 consecutive cytoplasmic domains (C1a and C1b)

• transmembrane domain (M2)

• 2 Consecutive cytoplasmic domains (C2a and C2b)

Consective cytoplasmic domains (C1a and C2a)

• are 40% identical → allows for association

• form catalytic core → composed of cytoplasmic regions that catalyze ATP→ cAMP

C1a, C1b, and C2a all bind

• regulatory molecules

cAMP

• polar, freely diffusing second messenger

cAMP targets

• Protein Kinase A (PKA, cAMP-dependent protein kinase or cAPK)

Protein Kinase A

• inactive PKA heterotetramer with

  • 2 regulatory AND 2 catalytic subunits

cAMP binds to the regulatory subunit

• 2 cAMP/ regulatory subunits

• 4 cAMP total can be bound

• dissociation of active catalytic monomers

Intracellular concentration of cAMP determines

• fraction of PKA in its active form

  • rate at which it phosphorylates its substrates

R subunit of Protein Kinase A

• competitively inhibits its C subunit

• contains 2 homologous cAMP binding domains (A and B)

• an auto inhibitor segment (resembles the C subunits substrate)

Inactive R2C2 complex

• autoinhibitor segment binds in the C subunit’s active sit which blocks substrate binding

• if cAMP is absent than can also inhibit since each R subunit requires 2 cAMPs

when B domain lacks bound cAMP

• masks the A domain

• prevents A domain from binding cAMP

Binding of cAMP to the B domain triggers

• massive conformational change

  • A domain can bind cAMP

A domain bound to cAMP leads to

• release of a now-active C subunits from the complex

cAMP activates

• protein kinase A (PKA; R2C2) by binding the regulatory dimer as R2 *cAMP4

Catalytic Subunit C dissociates

• R2 + cAMP4 + 2C

• activates various cellular proteins by catalyzing their phosphorylation

Signaling is limited by

• phosphotases action AND

• cAMP phosphodiesterase

Phosphodiesterases limit second messenger activity

• chemically based signaling system

signal molecule must eventually be limited to

• control the amplitude and duration of the signal

• prevent interference with the reception of subsequent signal

cAMP phosphodiesterase

cAMP is hydrolyzed to AMP by enzymes known as c-AMP phosphodiesterase

to decrease levels of cAMP

PDE superfamily includes

• cAMP phosphodiesterase and cGMP phosphodiesterase (guanine analog of cAMP

• encoded by at least 20 different genes grouped into 12 families