The Role of the V2 Vasopressin Receptor in Nephrogenic Diabetes Insipidus
by Sonia Prasad
In the past few years, a number
of inherited disorders have been traced to defects in both cell-surface
receptors and heterotrimeric G proteins.
Nephrogenic diabetes insipidus (NDI) is one of these disorders6. NDI is a rare kidney disease in which the
kidney tubules do not reabsorb adequate amounts of water, resulting in
excessive thirst and the passage of large quantities of dilute urine. The disorder is characterized by the
kidneys’ inability to respond to the antidiuretic hormone, arginine vasopressin
(AVP). NDI may be acquired at any time
during one’s life, or it may be inherited as an autosomal dominant, autosomal
recessive, or X-linked condition.
X-linked NDI is the most common form of inherited NDI – about 90% of the
cases are X-linked – and it is predominantly found in males. The X-linked condition is the result of
mutations in the V2 vasopressin receptor, which is responsible for
mediating the effect of AVP in the kidneys8. In order to understand this, it is necessary
to take an in depth look at vasopressin receptors and see how they operate.
Vasopressin is a peptide
hormone produced by the hypothalamus in the brain and secreted from the
posterior pituitary lobe3.
The effects of the hormone are regulated by vasopressin receptors. These receptors are made up of three
receptor subunits, which are all heptahelical membrane proteins: the V1a
receptor, the V1b receptor, and the V2 receptor. These receptors are found in the liver,
kidney tubule cells, platelets, smooth muscle vascular cells, and the central
nervous system, but the specific function of the V1a receptor in
these tissues is not known. The V1b
receptor, on the other hand, is known to stimulate the release of a hormone
called adrenocorticotropin, which stimulates the release of other
hormones. Together, however, the V1a
and V1b receptors regulate phospholipase activity, and the V2
receptor regulates adenylyl cyclase (AC) activity. These regulated activities are, in turn, involved in maintaining
fluid homeostasis and other cellular processes1.
Osmoregulation is the main
function of vasopressin receptors.
Osmoreceptor cells in the hypothalamus monitor blood osmolarity,
stimulating AVP release when blood osmolarity is higher than normal (300 mosm/L
in humans), and inhibiting release when osmolarity is lower than normal. For example, excessive water loss due to
sweating or diarrhea may cause an increase in blood osmolarity, thereby
stimulating AVP release. The main
targets of vasopressin are the distal tubules and the collecting ducts of the
kidneys, where the hormone increases the permeability of the epithelial cells
to water. This allows for increased
water absorption, preventing any further increase in blood osmolarity. Additional water intake will bring blood
osmolarity back down to normal. By
negative feedback, the subsiding osmolarity reduces the activity of the
osmoreceptor cells in the hypothalamus, resulting in less secretion of
vasopressin. Conversely, when large
volumes of water reduce blood osmolarity, very little (if any) AVP is released. The kidney is not as permeable to water so
less water is absorbed, resulting in an increased discharge of dilute urine3.
The way in which vasopressin
receptors work is similar to the way all other G-protein coupled serpentine
receptors work (see figure 1)1.
The system is activated when vasopressin binds to the receptors. As stated earlier, excessive water loss
triggers the release of AVP, which activates the system. When AVP binds to the V2
receptor, the heterotrimeric stimulatory G protein (Gs) is converted
to its active form when the bound GDP is displaced by GTP. In addition, the Gs protein
dissociates into Gsa and bg subunits. The Gsa subunit moves to AC,
stimulating its activity. The activated
AC then catalyzes the formation of cAMP from ATP, which activates protein
kinase A (PKA). Active PKA then allows for
the insertion of the aquaporin-2 (AQP-2) water channel into the lumenal surface
of the cell. Although the mechanism by
which PKA does this is still not clear, PKA probably phosphorylates some
protein critical to the process9.
The AQP-2 channel is crucial in regulating the kidney tubules’
permeability to water, and thus maintaining fluid homeostasis. In addition, the bg subunit may be involved in
the stimulation of phospholipase Cb (PLCb) activity.
Meanwhile, when
vasopressin binds to theV1 receptors, a similar reaction occurs with
a G protein – this time called a Gq protein. However, the reactions that occur when vasopressin
binds to the V1 receptors affects the cell’s metabolic processes
instead of water homeostasis. Again,
GDP is displaced by GTP, and the Gq protein dissociates into Gqa and bg subunits. The Gqa subunit (as well as the bg subunit) stimulates PLCb activity. This catalyzes the hydrolysis of
phosphatidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol (DAG)
and inositol-1,4,5-triphosphate (IP3). DAG remains associated with the plasma membrane while IP3 stimulates
the IP3 receptor located in the membrane of the endoplasmic
reticulum. This, in turn, stimulates
the release of Ca2+ from intracellular stores, and the Ca2+
leaves the cell (through a Ca2+ pump) and regulates other
enzymes. The release of Ca2+ from
the intracellular stores activates channels (called trp) which allow for the influx of extracellular Ca2+ to
maintain the Ca2+ balance1. Also, the release of Ca2+ activates protein kinase C
(PKC). PKC then catalyzes the transfer
of a phosphoryl group from ATP to a specific residue in one or more proteins,
regulating other enzymes9.
Not surprisingly, AVP has
the ability to reduce the response of the vasopressin receptors. Desensitization of the receptors has been
observed in transfected cells by studying the availability of the genes that code
for the receptors. V1a
desensitization is fairly quick and involves the sequestration of the receptors
inside the cell in tissues and transfected cells. Desensitization of this receptor is triggered by an agonist
called angiotensin II. Angiotensin II
is a powerful vasopressor that comes from the cleavage of a protein from the
liver. By its vasopressor action,
angiotensin II raises blood pressure and diminishes fluid loss by restricting
blood flow11. After the
agonist binds, AVP promotes the phosphorylation of the V1a receptor,
catalyzed by G protein coupled receptor kinases and PKC. Then, after being exposed to AVP, the
phosphates are quickly removed and the receptor is no longer present at the
surface of the cell, leaving the receptor desensitized. The ligand is then removed, and the V1a
receptor returns to the surface ready to receive another signal1.
Desensitization of the V2
receptor, on the other hand, takes longer and the receptor does not return to
the surface of the cell. The V2
receptor phosphorylation is catalyzed by G protein coupled receptor kinases
only. Unlike the V1a
receptor, the phosphates on the V2 receptor remain associated with
the protein for an extended period of time after exposure to AVP. Because of this, the V2 receptor remains
inside the cell. This definitely poses
a problem because it creates a reduction in the number of AVP binding sites,
which may affect osmoregulation1.
Since the V2
receptor is critical to fluid homeostasis, mutations in the receptor are of
great consequence, especially in relation to X-linked diabetes insipidus. The V2 receptor DNA encodes a
protein of 371 amino acids. Over 100
different mutations have been discovered on those amino acids in the
intracellular and extracellular loops, as well as on the seven transmembrane
domains of the protein2. In
most cases, V2 receptor mutations interfere with protein synthesis
and result in a reduced number or complete lack of receptors on the cell
surface. These mutations are most often
single amino acid changes, called missense mutations7. These variations in the primary structure of
the protein result in incorrectly formed secondary and tertiary
structures. Several of the mutations
that reduce or deplete the receptors at the plasma membrane are substitutions
of different amino acids in the second extracellular loop of the protein by
cysteine. Because cysteine has a
sulfhydryl group, it is capable of forming disulfide bridges with other
cysteine residues. Therefore,
inappropriate disulfide bridges may be formed between the new cysteines from
mutations and the preexisting cysteines already present in the protein. This would lead to the stabilization of the
wrong conformation of the protein, which would lead to a reduction in the
receptor’s binding affinity for AVP.
Decreased affinity for AVP and fewer receptors at the surface of the
cell leave the kidneys virtually unresponsive to circulating concentrations of
AVP. Several experiments have been
conducted to study the effects of V2 receptor mutations. In vivo,
the receptors are inactive, whereas in
vitro, Gs coupling and AVP binding affinity are greatly reduced2.
Perhaps the most widely
studied missense mutations occur on amino acid residues 204, 205, and 206 in
the C-terminal part of the second extracellular loop of the receptor (see
figure 2)11. Each mutation
is caused by a single base substitution in the second position of the
codon. The transversion of the bases C
and A results in threonine being replaced by asparagine at position 204
(T204N). The transversion of A and G
results in tyrosine being replaced by cysteine at position 205 (Y205C), and the
transition of T and A results in valine being replaced by aspartic acid at
position 206 (V206D).
Again, in vitro experiments were conducted to
study the effects of the mutations by way of transfection of two different cell
lines that are cultured specifically for transfection10. The two cell lines used were COS cells and
HEK-T cells. COS cells are Simian
fibroblasts, known as CV-1 cells, that are transformed by a virus, known
as SV40, deficient in the origin of replication4. HEK-T cells are human embryonic
kidney cells that expressed the T antigen. Higher amounts of mutant V2
receptor DNA (relative to the wild-type receptor DNA) were used in transfection
in order to ensure a sufficient expression rate of the mutant type. Each of the mutations (T204N, Y205C, and
V206D) were introduced separately to the wild type V2 receptor
DNA. Both the wild type and the mutants
were cloned into vectors which were used in transfecting the COS cells. The expression of each vector was then
determined using dose-dependent binding experiments with labeled vasopressin ([3H]AVP)
on membranes isolated from transfected cells or on intact cells. It was found that the V206D and Y205C
mutants were expressed in the COS cells but showed no binding of [3H]AVP
on the membranes or intact cells. These
mutations apparently reduced the binding affinities of the mutant receptors for
vasopressin. In addition, the T204N
mutant showed reduced binding affinity as well as reduced expression in the
cell. These experiments were then
repeated using HEK-T cells.
Interestingly, it was found that only cells expressing either the wild
type V2 receptor or the T204N mutant were able to bind AVP10,12.
Another series of
experiments was performed in order to test the mutants’ abilities to activate
adenylyl cyclase using the COS cells with AVP.
When AVP was introduced to the V206D mutant, cAMP levels were only 20% of
what they would normally be. When AVP was introduced to the T204N mutant, cAMP
levels were 55% of what they would normally be, but the levels increased with a
higher concentration of AVP. The Y205C
mutant with AVP, on the other hand, showed no significant levels of cAMP12.
In compiling the results of
these and various other experiments, it is plain to see that single base
mutations in different regions of the V2 receptor greatly affect the
receptor system. The replacement of
threonine by asparagine yields a receptor with reduced cell-surface expression
and reduced binding affinity for AVP.
At the same time, however, it is still able to stimulate AC, and thus
cAMP production, with the presence of high AVP concentrations. The replacement of tyrosine by cysteine
yields a receptor that is nearly
inactive, with a very low affinity for AVP and impaired cAMP production. Finally, the replacement of valine by
aspartic acid yields a receptor with a low binding affinity for AVP and very
little cAMP production. Overall, it has
been found that both the T204N and V206D mutant receptors are involved in or
cause nephrogenic diabetes insipidus.
Clearly, if there is a
problem with the vasopressin receptors – particularly with V2
receptor – kidney function will be affected.
Since vasopressin is essential in regulating fluid retention, mutations
in the receptors can cause serious disorders.
If the receptor is not expressed at the surface of the cell, AVP cannot
bind, and the signaling cascade does not function. If the receptor has a low affinity for AVP, the signaling cascade
may not work efficiently. If the
receptor undergoes a conformational change, AVP may not bind at all, and again,
the signaling cascade will not function.
If there is a problem activating adenylyl cyclase due to the Gs
protein – as is the case with some missense mutations – the cascade will not
proceed. In each of these situations,
the important thing to remember is that vasopressin must bind to its
receptor. Furthermore, AC activation
must catalyze the production of cAMP from ATP in order to stimulate PKA so that
the AQP-2 channel can be inserted into the membrane. As stated earlier, the AQP-2 channel plays an important role in
regulating the kidney tubules’ permeability to water, and thus regulating fluid
homeostasis. Without the channel, water
would have no way of being reabsorbed by the kidneys. This is what causes large amounts of dilute urine to be excreted,
leaving the body in danger of dehydration.
Although NDI is not a disorder that affects a great number of people –
at the most 1 in 100,000 and usually males8 – treatment is
important. Based on the way this
disorder comes about, one may suggest treatment with antidiuretic drugs. However, it is important to keep in mind
that the success of such drugs is limited by the type of V2 receptor
mutation, if in fact there is a V2 receptor mutation. If not diagnosed promptly, NDI may cause
chronic dehydration, which could lead to mental retardation, improper growth
and even death.
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