3140 - G proteins

4 Gα subfamilies

• Gαs

  • Has 3 splice variants 

• Gαi/o

  • Largest number of splice variants (9)

• Gαq

  • 4 splice variants

• Gα12/13

  • 2 splice variants 

how many Gβ genes?

• Gβ1 - Gβ4 have similar properties 

• Gβ5 has different structure and does not interact well with Gγ

what is special about Gβ5?

• has different structure and does not interact well with Gγ

how many Gγ genes?

12

Gαs roles:

• Activates adenylyl cyclase (AC)

• Important in asymmetric cell division during development 

Gαi roles:

• Inhibits adenylyl cyclase and activates phosphodiesterase 

• Gαq roles:

• Activates PLC => activates DAG and IP3

• Activates RhoGEF

Gα12/13 roles:

• Activates multiple RhoGEFs

  • Stimulates dissociation of GDP from Rho proteins 

• Gβγ roles:

• Regulates AC, PLC, VG Ca channels, K rectifier channels

• Active only when associated with active Gα subunit 

• Gα is _____ when bound to GDP

inactive

how does GDP activate Gα?

• GDP dissociates

  • Process is accelerated by GPCRs to help GDP get replaced faster

• GTP binds because it is present at 10x higher concentration than GDP

  • Gα becomes activated 

GTP is how much more abundant than GDP

10x higher concentration

GAP proteins

hydrolyzes GTP

how is Gα inactivated?

• Signaling lifetime is as long as GTP remains bound to alpha subunit 

  • GTP eventually gets hydrolyzed, process is accelerated by GAP effector proteins 

how is Gαs expressed?

• biallelically (one copy from each parent)

Whole body knockout or activating mutation of Gαs is:

embryonic lethal

what happens in Gαs mutations?

decrease GTPase activity, causing G proteins to remain in GTP bound state for longer and spontaneous GPCR signaling

McCune Albright Syndrome 

Gαs mutation in which there is mosaic distribution of affected tissues (mixture of cells with and without mutation)

symptoms of McCune Albright Syndrome

• Endocrine dysfunction 

• Secondary hyperparathyroidism 

• Fragile malformed bones 

  • Fibrous dysplasia 

    • Scar-like tissue grows in place of normal bone, leading to bone deformity, brittle bones

• Hyperpigmented skin patches 

  • Upregulated melanocyte stimulating hormone 

• Early puberty 

Pseudohypoparathyroidism (PHP)

• Gαs mutation

• PTH present but absent signaling 

Albright Hereditary Osteodystrophy 

a form of Pseudohypoparathyroidism (PHP)

• Ossification, short stature, brachydactyly (deformed digits), obesity, cognitive impairment 

symptoms of Pseudohypoparathyroidism (PHP)

• Hypocalcemia

• Hyperphosphatemia 

Fibrous dysplasia 

• Scar-like tissue grows in place of normal bone, leading to bone deformity, brittle bones

mutations in Gαs are usually due to loss of the ___ gene

paternal

PHP1A

• Maternally inherited inactivating Gαs mutation 

  Paternal gene is normal 

Mutations in maternal gene have ____ effects than mutation in the paternal gene 

worse

PHP1A results in:

• Loss of PTH receptor in kidney => kidney can’t reabsorb Ca

• Elevated PTH secretion 

Pseudopseudohypoparathyroidism (PPHP)

• Paternally inherited inactivating Gαs mutation 

  • Maternal gene is normal => less serious defects

symptoms of PPHP

• Smaller size at birth 

• Symptoms less severe than PHP1A

  • Normal PTH, Ca, and phosphate levels 

Gαs A366S causes:

• Both Gαs alleles mutated 

• Causes increased GDP dissociation rate => constantly active state

Gαs A366S symptoms:

• Thermolabile activation 

  • G protein denature more easily 

• Similar symptoms as PHP1A

• Mutant survives in Leydig cells (testosterone production), causing increased LH signaling in males and early puberty 

Adenylyl cyclase function

• Primary activating target of Gαs

• Converts ATP to cAMP 

  • cAMP binds to many targets to alter cell behaviour 

AC structure:

• Alternating hydrophobic and hydrophilic domains 

AC hydrophobic domain has ____ transmembrane helices

6

which region of AC performs catalytic function?

hydrophilic regions

AC is activated by:

Gαs

AC is inhibited by:

Gαi

Gβγ effect on AC?

• Activates or inhibits AC

Ca ____ AC

inhibits

Calmodulin _____ AC

activates

PKA _____ AC

inhibits

effect of PKC on AC:

• Activates or inhibits AC

how many isoforms of AC?

9

PKA is activated by:

cAMP

PKA structure:

•  heterotetrameric complex with  2 catalytic subunits held inactive by a regulatory subunit dimer

what are the two types of PKA and where are they found?

• Type I

  • Predominantly cytoplasmic 

• Type II

  • Associates with specific organelles

  • Localizes to specific areas of cell with A kinase anchoring proteins (AKAPs)

how many cAMP bind to PKA?

2 cAMP molecules binds to each regulatory subunit, (4 cAMP binding in total)

how does cAMP binding activate PKA?

binding leads to conformational change that releases the catalytic subunits

PKA phosphorylates receptors, ion channels, enzymes, CREB

CREB

• Transcription factor activated by PKA that when phosphorylated, enters nucleus and binds to DNA binding elements CRE to regulate gene transcription 

cAMP also activates:

CNG ion channels in pacemaker cells of heart

Epac

Epac

exchange protein activated by cAMP

• Activates Ras-like small GTP binding proteins Rap1 and Rap2

• Promotes cAMP dependent exocytosis 

PDE

phosphodiesterase

decreases cAMP or cGMP levels

cAMP selective PDE:

PDE2a, PDE3, PDE4, PDE7, PDE8, PDE10a

cGMP selective PDE:

PDE1a, PDE1b, PDE5a, PDE6, PDE9a

PDE1 is activated by _____ to _____ cAMP levels

calmodulin

decrease

PKA increases activity of which PDE?

PDE4d

PKA and PKG increase activity of which PDE?

PDE5

pertussis toxin (PTX)

inactivates Gαi/o covalently modifying residues that cause G protein to be stuck in inactive GDP form 

pathways lots with PTX treatment:

• Suppression of Ca channel opening 

• Activation of rectifying K channels 

• Activation of PLC beta

Transducin (Gαt)

Special member of Gαi/o family specific to the visual system

how is Transducin (Gαt) activated?

• Activated by activation of rhodopsin in presence of light 

Transducin (Gαt) pathway

• in presence of light, rhodopsin activates transducin

• Activates PDE6, a cGMP selective PDE

  • Leads to reduction of cGMP concentration, closing cGMP gated cation channels to hyperpolarize cell

activators of Rho GTPase:

• Gα12/13

• Gαq/11 

  • Activates p63RhoGEF

• Gβγ 

  • Can activate GEFs for small G proteins 

 PLCβ is activated by:

Gαq

Activation of PLCβ causes:

• hydrolyzation of PIP2 into 2 second messengers: 

  • DAG

  • IP3

DAG

2nd messenger that activates PKC

IP3

• Promotes release of Ca from SR/ER to raise cytosolic Ca levels 

• Ca activates some PKC isoforms

ways that G proteins affect intracellular Ca:

• IP3 binds to ER/SR receptors to release Ca

• CNG channels permit Ca influx into retinal, olfactory, and pacemaker cells 

• PKA phosphorylated L type Ca channels to increase intracellular Ca

• Gβγ inhibits VG Ca channels 

how is Ca rapidly removed from the cytosol?

• Na/Ca exchanger pumps help remove Ca from cytosol 

• SERCA pumps (Ca ATPase) return Ca to SR

• PMCA removes Ca from cell 

how is CAM Kinase II is activated?

• by Ca bound calmodulin 

• Autophosphorylates to prolong duration of activation 

memory switch of CAM kinase II

Kinase still remains active even after cytosolic Ca decreases

CAM kinase II is inactivated by:

phosphatase removes its phosphate 

what is calmodulin’s role in smooth muscle contraction?

• Calmodulin activates myosin LC kinase, which phosphorylates myosin light chain to facilitate actin and myosin interaction 

calmodulin’s role in blood vessel relaxation:

• Activation of calmodulin activates eNOS

• eNOS produces NO

• NO enters smooth muscle cell and activates guanylyl cyclase 

• Guanylyl cyclase activates PKG 

  • Decreases activity of VG Ca channel

  • Decreases contraction