G protein alpha subunit in its GTPbound form, highlighting amino acids changed by point mutations th

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G protein Mutations Causing Disease
G protein alpha subunit in its GTP-bound form,
highlighting amino acids changed by point
mutations that cause human endocrine diseases.
Mutational replacements of red residues impair
GTP hydrolysis these sites are mutated in growth
hormone secreting tumors of the pituitary.
Replacement of either cyan residue produces an
inactive G protein alpha subunit, causing
pseudohypoparathyroidism. Bound nucleotide is
light green.
from Henry Bourne lab page
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gsp mutations of Gas Gs alpha- single
gene Point mutations in the alpha subunit
R201C, R201H, Q227R, Q227L Mutations inhibit
GTPase activity of G protein, resulting in
constitutively active Gas subunit. gsp mutations
are seen in tumors from cells where cAMP
stimulates growth. gsp mutations occur in 40 of
growth hormone-secreting pituitary adenomas and
30 of thyroid hyperfunctioning adenomas. Other
G protein subunits and disease--Gi alpha?????
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McCune-Albright Syndrome. Somatic mutation of
Gs alpha early in development Effects of
activating MSH and gonadotrophin receptors
evident.
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Pseudohypoparathyroidism. One inactive copy of
Gs alpha. Resistant to PTH, TSH, ACTH, GHRH (and
others) Complex tissue-specific genetic
imprinting syndrome worse if inherit bad
copy from mom than from dad
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Testotoxicosis
Symptoms Precocious puberty indicating
premature testicular activation (normally
testosterone production is stimulated by LH, a
GPCR coupled to cAMP formation) Other findings
looked like pseudohypoparathyroidism, i.e.
impaired responses to PTH, TSH causing PTH and
thyroid abnormalities (due to a defect in Gs
alpha)
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Testotoxicosis
Conclusion Syndrome due to a temperature-sensiti
ve Gs alpha Protein inactive at 37 but
constitutively active at the lower temperature
of the testes, 32
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Signal Transduction in the Retina
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Effect of RGS Mutation
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Visual Defect with R9AP Mutation
R9AP is a membrane anchor for RGS9/Gbeta5 dimer
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Visual Defect with R9AP Mutation
Rods RodsCones
Cones
Defect in second response similar to that in RGSo
KO mice
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Clinical syndrome Hypothesis Sequence
candidate gene from affected vs. unaffected
individuals Construct mammalian expression
vectors for WT and mutant genes usually use a
strong constitutive promoter like CMV Transfect
cells, often COS, CHO or HEK293 Study protein
signaling, localization with and without
activation Constitutively active mutations most
commonly identified X-linked are exceptions
Typical Approach for Detecting Receptor Mutations
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TSH Signal Transduction
Gas
Aden Cyclase
Gbg
TSH Receptor
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Signaling by Mutant TSH Receptors
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Mutations in TSH Receptors
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Typical Behavior of Constitutively Active
Receptors
Constitutively Active Receptor
SECOND MESSENGER
Inactive receptor
AGONIST
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Graves Disease
Thyroid stimulating Abs
TSH Receptor
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Graves Disease Antibodies are formed against
TSH receptor Antibodies activate receptor
increase cAMP in vitro increase thyroid
hormone production increase thyroid cell
growth (goiter) Antibodies recognize many
different regions on extracellular surface of
receptor
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TSH Receptor
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Mutation Altering TSH Receptor Specificity
During pregnancy, when hCG is very high, woman
became severely hyperthyroid due to excessive
stimulation of thyroid through mutant TSH receptor
LH receptor
Normal TSH receptor
Mutant TSH receptor
cAMP
cAMP
Normal TSH receptor
TSH
hCG, placental hormone
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Some forms caused by mutations in rhodopsinMany
autosomal dominant forms are due to defects in
receptor trafficking Rhodopsin
synthesized Cannot get to surface ? Basis for
dominance
Retinitis Pigmentosa
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Visual GPCRs
Rhodopsin
7 aa differences
Extinction Coefficient
500
410
532
563
400
600
Wavelength, nm
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Red and green opsins are on X chromosomeBlue
opsin and rhodopin are not on the XRed/green
opsin genes are prone to unequal crossing over,
hence color blindness is common.
Visual GPCRs
RG- red-blind
RG normal
anomalous trichromat
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MSH Receptor Family
Melanocortin Receptors 1-5 MC1MSH receptor
(pigmentation) MC2ACTH receptor
(adrenocorticotrophic hormone) MC3central
nervous system MSH receptor MC4central nervous
system MSH receptor MC5exocrine
glands Mutations in MSH receptor-coat
coloration Mutations in MCR4-severe
obesity Unique aspect Both agonists (MSH) and
antagonists (agouti, agouti-related peptide)
control activity of MSH receptor, in some
animals, and MCR4, in all species
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MSH Receptor Variants
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Effect of Agouti Protein
Agouti acts as classical competitive antagonist
at MSH receptor and MC4 receptors, not other
receptors in this family
MSH Receptor and MC4 Receptor
MC3 and MC5 Receptors
Agouti
Agouti
cAMP
cAMP
MSH
MSH
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Agouti Coloration
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Agouti Overexpression Blocks MC4 Receptors
Activation of MC-4 receptor normally inhibits
NP-Y Mutation causes ectopic production of
agouti peptide (MC-4 receptor antagonist) Decrease
d activation of MC-4 receptor in
hypothalamus Increased NP-Y, potent orexigenic
peptide Hyperphagia and weight gain
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Effect of Mutation in MC4 Receptor
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