G-proteins

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Biol 456
Comparative Vertebrate
Endocrinology
Instructor: Dr. Robert Harris
Office: 2530 Biological Sciences
Phone: 822-5709
Email: [email protected]
Course requirements:
Texts:
Principles of Animal Physiology 2nd ed.
Moyes and Schulte
Endocrinology 5th ed.
Mac E. Hadley (recommended)
or
Vertebrate Endocrinology 3rd ed.
David O. Norris (acceptable)
Mark Breakdown
Term Paper:
Midterm Exam:
Final Exam:
50%
30%
20%
Course Total:
100%

Endocrine signaling is probably the oldest
control mechanism.

Many organisms get by quite nicely without any
kind of nervous system.

Probably arose as mechanism for maintaining
stable internal environments.

Endocrine signaling is probably the oldest
control mechanism.

Many organisms get by quite nicely without any
kind of nervous system.

Probably arose as mechanism for maintaining
stable internal environments.
Definitions:



Broadly speaking, a hormone is a chemical that
is produced by one specific cell type, secreted
into the blood or interstitial fluid, and has
specific effects on a different cell type(s).
Secreting cells are usually clumped together to
form a discreet gland.
There are two types of glands:
Endocrine
 Exocrine

Features of Hormones:



Found in specialized cell types
These cells must synthesize the compound (or
precursor) specifically for signaling.
Cells must secrete the compound (or precursor)
in response to a specific stimulii.
Features of Hormones II:



There must be a mechanism for clearance of the
compound from the blood, or interstitial fluid.
Drugs that affect the hormone will also affect
the hormonal response in the target tissue.
Exogenously applied compound must be
chemically identical to the hormone and must
mimic the biological effects of the endogenous
compound.
MODES OF HORMONE DELIVERY I:

AUTOCRINE:


Hormone released feeds-back on the cell of
origin, again without entering blood circulation.
PARACRINE:
Hormone released diffuses to its target cells
through immediate extracellular space.
 Blood is not directly involved in the delivery.

MODES OF HORMONE DELIVERY II:

ENDOCRINE:


Most common (classical) mode, hormones delivered
to target cells by blood.
NEUROENDOCRINE:

Hormone is produced and released by a neuron,
delivered to target cells by blood.
HORMONE-TARGET CELL
SPECIFICITY

Only target cells, or cells that have specific
receptors, will respond to the hormone’s
presence.

The strength of this response will depend on:
Blood levels of the hormone
 The relative numbers of receptors for that hormone on
or in the target cells
 The affinity (or strength of interactions) of the hormone
and the receptor.

Major endocrine glands in the body
HALF-LIFE, ONSET, and
DURATION of HORMONE
ACTIVITY


The affinity of hormones to their specific
receptors is typically very high
The actual concentration of a circulating
hormone in blood at any time reflects:
Its rate of release.
 The speed of its inactivation and removal from the
body.


The half-life is the time required for the hormone to
loose half of its original effectiveness (or drop to half
of its original concentration.

The time required for hormone effects to take place
varies greatly, from almost immediate responses to
hours or even days.

In addition, some hormones are produced in an
inactive form and must be activated in the target cells
before exerting cellular responses.

In terms of the duration of hormone action, it ranges
from about 20 minutes to several hours, depending on
the hormone.
Considerations when looking for
hormonal responses:

Tissue sensitivity may change in response to
other hormones (priming).

Effects of photoperiod.

Effects of temperature.
Basic Techniques

Extirpation/replacement (classical technique).
Hormonal source is removed or ablated, to see if the
response is abolished.
 Once response has been abolished, source tissue or
exogenous hormone is replaced in an attempt to
restore response.

Injection of hormone
 Allograft
 Injection of live dissociated gland cells


Establish the physiological range of hormone
concentrations.



Usually conducted in conjunction with extirpation
experiments.
Endogenous source is removed and known concentrations or
doses administered to determine the threshold and maximal
doses for the hormone.
Other considerations:


Dose-response curve may be biphasic and not sigmoidal.
You must have 8-10 individual animals at each dosage to
make results significant.

Radioimmunoassay (RIA):

Used to measure hormone concentration.

Uses antibodies specific to the hormone.

Also uses radiolabelled purified hormone.

Must create a standard curve (for competitive
binding)

Drawbacks:

No guarantee that antibodies are recognizing the
biologically active form of hormone.


May be measuring inactive fragments.
Often get cross-reactivity with other hormones.

Closely related hormones have significant sequence
homology.

Immunoradiometric assay (IMRA):

Also uses antibodies.

Uses two antibodies, one against C-terminus and one
against N-terminus.
One AB is bound to a support matrix (usually
avidin-coated beads).
 Other AB is conjugated to an indicator of some
sort.


Could be radiolabel or fluorescent label.
Measures virtually all the hormone in the sample.
 Only measures intact hormone.


i.e. both end must be present.

High performance liquid chromatography (HPLC):
Can be used to separate out different hormones.
 Like any biochemical purification protocol, it can be
used to isolate a specific hormone.
 Different hormones will have different migration rates
on separation medium.
 Can be used to track metabolites of a hormone.
 Needs a great deal of characterization using purified
hormone.
 FPLC is a similar technique.


Immunocytochemistry:
Antibody based and uses two antibodies.
 First AB recognizes the hormone.
 Second AB recognizes the first AB (must come from
different species).
 Second AB is labeled.
 Can be used to localize cells secreting hormone.



Take tissue section, fix, permeabilize and label.
Note: All the problems associated with Abs apply.

Receptor binding assay (aka RRA):

Uses competitive binding between hot and cold
hormone, usually on the membrane fraction of
a cell homogenization.
Will provide the number of available binding
sites on target tissue.
Will also provide the binding efficiency.



Molecular techniques:
Currently very “sexy”.
 Usually transfect cell line with the gene for the
hormone or receptor.
 You can also measure mRNA levels (only for
peptide hormones).
 Must have at least a partial sequence.
 Increased hormone production may not be
associated with elevated mRNA levels.


mRNA may code for a processing enzyme.
CONTROL OF HORMONE RELEASE:

The synthesis and secretion of most hormones
are usually regulated by negative feedback
systems.


Organized into “loops”.
As hormone levels rise, they stimulate target
organ responses. These in turn, inhibit further
hormone release.

The stimuli that induce endocrine glands to
synthesize and release hormones belong to one
of the following major types:

Humoral

Chemical changes in the blood (eg. Ca2+ or glucose).
Neural
 Hormonal (as seen in feedback loops)

CHEMISTRY OF HORMONES


Peptide hormones: largest, most complex, and most
common hormones. Examples include insulin and
prolactin
Steroid hormones: lipid soluble molecules
synthesized from cholesterol. Examples include
gonadal steroids (e.g testosterone and estrogen) and
adrenocortical steroids (e.g. cortisol and
aldosterone).

Eicosanoids: small molecules synthesized from
fatty acid substrates (e.g. arachidonic acid)
located within cell membranes

Amines: small molecules derived from individual
amino acids. Include catecholamines (e.g.
epinephrine produced by the adrenal medulla),
and thyroid hormones.
Receptor Tyrosine Kinases
Figure 4.16
MAP kinases
• Activated RAS signals to
MAP-kinase-kinase-kinase
• MAPKKK signals to MAPkinase-kinase
• MAPKK signals to MAPkinase
• MAP-kinase phosphorylates
a variety of target proteins
Figure 4.17
Transforming growth factor – b (TGF-b) receptor
• An important
serine-theronine
kinase receptor
enzyme
• Involved in
regulating many
cellular processes
• Malfunctions
result in many
diseases
including Cancer,
atherosclerosis
etc.
Figure 4.18b
G-Protein-Coupled Receptors
Ligand binds to transmembrane receptor
 Receptor interacts with intracellular G-proteins



Named for their ability to bind guanosine nucleotides
Subunits of G-protein dissociate

Some subunits activate ion channels



Changes in membrane potential
Changes in intracellular ion concentrations
Some subunits activate amplifier enzymes

Formation of second messengers
G-proteins coupled receptors (GPCRs)
•
•
•
•
Large protein family (over 1000 members)
Seven-transmembrane domain proteins
Interact with a trimeric intracellular protein termed “G-protein”
(guanine nucleotide binding protein)
G P ro t e in C o u p le d R e c e p to r
F rom : B oron & B ou lpa ep
M ed ic a l P hys iolog y, 2003, F ig.4 -3
G-Protein-Coupled Receptors
Figure 3.25
G-proteins
S tru c tu re o f h e te ro trim eric G p ro te in s
L inked directly to the plasm a
m em brane via a lipid anchor
G-protein
a, b and g
subunits
There are many types of G proteins – 3 major families
Ta b le 1 5 -3 . Th re e M a jo r F a m ilie s o f Trim e ric G P ro te in s *
F A M IL Y
S O M E F A M IL Y M E M B E R S
I
Gs
a
activ ates ad en ylyl cy clase;
activ ates C a 2 + ch ann els
G o lf
a
activ ates ad en ylyl cy clase
in o lfacto ry senso ry
neuro n s
Gi
a
in hib its ad en ylyl cy clase
II
Go
A C T IO N M E D I A T E D B Y
bg
activ ates K + chan nels
bg
activ ates K + chan nels;
in activ ates C a 2 + ch an n els
a an d b g
III
FU N C TIO N S
activ ates p ho sp ho lip ase C b
G t (tran sdu cin )
a
activ ates cyclic G M P
p ho sp ho d iesterase in
verteb rate ro d
p ho to recep to rs
Gq
a
activ ates p ho sp ho lip ase C b
Fam ilies are d eterm in ed b y am ino acid seq u en ce related n ess o f t he a sub units. O nly selected ex am p les are sho w n.
A b o ut 2 0 a sub u nits and at least 4 b sub units and 7 g sub u nits h ave b een d escrib ed in m am m als.
*
Second Messengers
Table 3.3
Cyclic-AMP Signaling
Figure 3.27
Im p ortan t T ools for stu d y in g G s/G i
C h olera T oxin – A D P -ribosylates G s. T his locks G s in the G T P bound conform ation, lead ing to constitu tive ac tiva tion o f aden ylyl
c yc lase, and accord ingly, inc reased cA M P production.
P ertu ssis T oxin – A D P -ribo sylates G i. T h is locks G i in the inac tive
G D P -bound confo rm a tion. S ince activated G i has an in hibitory e ffec t
on aden ylyl c yc lase, pertussis toxin po tentiates cA M P accu m ula tion.
T hese tw o toxins a re used to determ ine w hich type of G prote in a
recepto r is signaling th rough .
Inositol-Phospholipid Signaling
Figure 3.26

Lecture Notes and synopsis are posted at:
http://www.zoology.ubc.ca/~harris/
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