COX Receptors and GPCRs as Model Systems for Studying
Effect of Structure on Specificity
Abstract
COX 1 and 2 receptors are involved in the production of essential
prostaglandins that maintain the gastrointestinal lining. The structural
difference between COX 1 and COX 2 is minute enough to serve as an example of
acute substrate specificity. On the other hand, G-protein coupled receptors
(GPCRs) are the gate keepers in intercellular communication. These receptors are
activated by the binding of a specific extracellular substrate. GPCRs consist of
a large superfamily and possess diverse structural differences to account for
the need for different substrates in order to prevent mixed signals in cellular
communication. By studying the structure of COX receptors and GPCRs, comparisons
can be made between the levels of substrate specificity. The structural
difference can be narrowed down in COX receptors to one amino acid; isoleucine
in COX-2 is substituted with valine in COX-1 at two locations at the active
site. Specificity of GPCRs is still not well elucidated. However, the structural
factors which affect GPCR specificity cannot be narrowed down to one amino acid
difference. Instead, it
results from a cooperation of
several
factors, such as intramolecular
interactions, covalent
modifications, and structural flexibility. Ultimately, COX receptors and
GPCRs help serve as model systems for studying the effect of a receptor's
structure on its specificity.
Introduction
Proteins are the molecular machines of life with
diverse
functions from sensing light to serving as a chemical bridge to allow
cells to communicate. Being multi-cellular organisms, humans have specialized
cells which require a higher level of intercellular communication to maintain
life. Of course, with such complex functions comes a myriad of signal molecules
and other substrates which react with various proteins within our cells. Protein
receptors are highly specialized to have varying degrees of specificity for
different
substrates. The substrate specificity of cyclooxygenase 1 and 2 varies
greatly from that of G-protein coupled
receptors and a comparative study of these two receptors can help clarify the
regulation of specificity by structure.
COX 1 and 2
The cyclooxygenase receptors are responsible for production of
prostaglandins (PGs) which are crucial to the maintenance of the
gastrointestinal lining and kidney function2. Cyclooxygenase 1 is
present in a constitutive form, and thus a steady amount of PGs necessary for
functions such as maintaining gastrointestinal lining are produced regularly.
Along with COX-1, a second type of cyclooxygenase receptors exists called COX-22.
Unlike that of COX-1, COX-2 activity is induced by pro-inflammatory proteins
known as cytokines, which are responsible for both nonspecific and specific
immune responses2. In the case of COX-2, the immune responses are
nonspecific such as inflammation.
Figure 1:
Basic crystallography showing the two dimerized monomers. Perioxidase site is
above the heme prosthetic group (shown in red) while the oxidation site is near
the membrane domain (shown in yellow)14.
Each monomer of COX receptors has two catalytic functions: an oxidizing
function, followed by a reduction function. The COX (oxidation) site is found on
the side of the monomer near the endoplasmic reticulum membrane while the POX
(catalytic unit called peroxidase for reduction) site is located on the opposite
end of the monomer4. As shown in Figure 2, the oxidation process
involves the addition of two oxygen molecules to the substrate, Arachidonic Acid
(AA), forming a peroxidase with a 5 carbon ring called PGG2
(Prostaglandin-G2)3. The POX site reduces PGG2
to PGH2 (Prostaglandin-H2) via the addition of two
hydrogen atoms. PGH2 is a precursor of many prostaglandins4.
Figure 2:
Oxidative and reductive catalytic functions of COX monomers3.
Based on research results, it has been suggested that only one monomer
can perform catalytic activity at any given time and substrate binding to one
monomer causes a conformational change in its partner monomer implying
reciprocal allosteric regulation between them4. It is possible that
the specificity for Arachidonic Acid (AA) may be regulated by this
conformational change.
Inhibition of COX-1 receptors can lead to deleterious side effects such
as gastrointestinal ulcers and bleeding due to the restriction of the passive
biosynthesis of necessary prostaglandins that maintain the gastrointestinal
lining and kidney function2. Prostaglandins are also mediators of
vasodilation because they serve as substrates to receptors that control muscle
constriction2. They promote relaxation of the blood vessels which
ultimately dilates them2. The dilation of the blood vessels
constitutes inflammation and swelling which is perceived as a painful sensation
by the individual. Many pharmacological benefits of pain killers can be improved
by selectively inhibiting COX-2 receptors because many of the NSAIDs
(non-steroidal anti-inflammatory drugs) in use such as aspirin inhibit both
COX-1 and COX-2. The deleterious side effects of the NSAIDs come from preventing
the steady biosynthesis of mandatory PGs by inhibiting COX-1 even though they
may effectively decrease the inflammation, swelling and the pain sensation by
inhibiting COX-2 simultaneously 2.
When COX-2 was discovered, it was found to be extremely identical with
COX-1 and a 63% homology existed between them. However, there were a few
differences in the amino acid sequences at the entrance to the active site and
one within the active site 12. Copying sequences from COX-1 and
overlaying them over COX-2
using molecular modeling techniques allowed a better understanding of which
amino acid differences lead to a substantial change in specificity2.
In this case, one amino acid within the active site was determined to be the
main factor in producing the difference in specificity between COX-1 and COX-21.
This determination was made by a molecular modeling experiment via point
mutagenesis (selectively replacing a specific amino acid with another)1.
It was found that COX-2 had the amino
acid valine at residues 434 and 523 whereas COX-1 had isoleucine at the same
locations2. As shown in Figure 3, valine's side chain is smaller with
a structure of R(CH)(CH3)2 whereas isoleucine's side chain
contains an additional -CH2 group ['R(CH)(CH3)(CH2)(CH3)]
2.
What does COX-2 gain from the valine substitution of isoleucine? It simply
allows for more space in the active
site.
Valine
Isoleucine
Figure 3:
3-D structures of valine and isoleucine.
In the absence of an extra -CH2 group, there is less steric hindrance
for the binding of an inhibitor molecule (e.g. NSAID) and thus a wider range of
inhibitors bind to COX-2 compared to COX-1. COX-2 specific NSAIDs are the target
of research and pharmacological interests. Overall, this small variation in a
single amino acid structure can lead to a big difference in drug selectivity
between COX-1 and COX-2 making the inhibition of inducible, pro-inflammatory PGs
synthesis possible without the inhibition of essential PGs synthesis. In the
pursuit of this goal, the new generation NSAIDs including Celecoxib and MK-966
that show no signs of gastrointestinal damage are synthesized. One study showed
that these COX-2 selective inhibitors helped relieve pain after dental surgery
in some volunteers2. Subsequent studies aim to test COX-2
selective inhibitors on colon cancer and Alzheimer's which are known to be two
contributors to inflammation2. SC-588 is another COX-2 selective
inhibitor found to help eliminate inflammatory effects and has shown no side
effects such as gastrointestinal damage1. Although the tested drugs
were used on select patients, several side-effects of COX-2 inhibitors have been
hypothesized2. One includes inhibition of cell growth and
repair since the same inflammatory cytokines are also associated with cells that
release growth factors2.
GPCRs
GPCRs constitute a large family of membrane proteins which are of
significant pharmacological value
since
almost 50% of the drugs on the market target these receptors and
development of new drugs would be facilitated with a better understanding of
their structure10. Being membrane proteins, GPCRs prove to be
difficult in structure determination. Part of this difficulty is due to their
complexity as well as how the membrane layer helps to maintain the delicate
structure of the GPCR5. GPCR structure makes it hard to
crystallize due to the conformational flexibility in its extracellular loops.
Furthermore, analysis of the crystal structure is typically not fully indicative
of how the proteins would function in
vivo since GPCRs are
dynamic and their structures shift to allow them to conform to a variety of
substrates5.
Figure 4 shows the universal structure of GPCRs consisting of
7-transmembrane helices (TMH), an active extracellular loop which binds to
signaling molecules, and an intracellular loop which serves as the signal
transducer
and interacts with an internal heterotrimeric G-protein9. Helices are
the most common structure found in the transmembrane space due to their
adaptation of hydrophobic surfaces which allow them to diffuse and exist in the
lipid bilayer9. Another property that unifies the superfamily of
GPCRs is the mechanical function of the helices. Due to the helices’ rigidity,
they are ideal for mechanical function serving as a rotator, a lever, or a
piston9. The highly dynamic extracellular loop undergoes structural
changes when it binds to a substrate and sends this conformational change down
through the helices which serve to relay the signal mechanically similar to
piston-like, lever-like, or rotational movement9. A combination of
movements such as the rotation of a curved or bent helix may be used to achieve
a large displacement at the intracellular end9.
Although the structure of GPCRs can be considered universal, they
constitute a very large family of proteins and have 6 subfamilies12.
Serving as a true display of its wide range of specificity, GPCRs within the
same subfamily may have sequence similarities up to 20% or higher and
dissimilarities as small as 3% which suggests a strong homology within the
subfamilies yet a wide range of diversity in terms of structure versus function12.
A comparative analysis of structural similarities within subfamilies was
conducted by Worth et al12, and the results clearly showed the
similarities within subfamilies even though each independent receptor still has
distinct functions such as light perception in rhodopsin or sympathetic nervous
system mediation12.
Figure 4:
Topology of Subfamily A GPCRs. Superimposition of 5 template structures12.
In addition to the seven trans-membrane helices, there is also an
intracellular eighth helix which plays a role in receptor activation by
G-protein receptor kinases (GRK)7. One example of the helix 8's
function can be seen in the throtropin-releasing hormone receptor (TRHR), a
class A GPCR, where a conserved positively charged site facilitates
phosphorylation (the addition of a phosphate group) by a kinase which in turn
activates the receptor. (Conversely, palmitoylation, addition of a fatty acid,
palmitate, occurs at the end of TMH 7 and serves to keep TRHR in its inactive,
non-constitutive form6.) Altogether, this is an example of how a
particular module found in one class of GPCRs may have a universal function
within that class, as in the case with helix 8 and phosphorylation7.
Function may vary due to the lack of conserved motifs between the classes of
GPCRs. To further exemplify similarities in structure regardless of large
differences in homology, research has been done to model class B GPCRs using
class A templates13. Little is known about class B GPCRs and thus
comparative studies between class A and class B are difficult due to the lack of
homology, however it is possible to identify the analogous motifs by
searching the specific locations in class B GPCRs based on the locations of
their analogs in class A13.
Discussion
COX receptors and GPCRs regulate their own levels of specificity and this
has been taken advantage of in pharmacology. GPCRs, the super family of membrane
proteins, are responsible for many processes of signal transduction and cellular
communication. The active loops of GPCRs are dynamically shifting structures and
some receptors have large active sites which can allow substrates with a wide
range of sizes and thus have a broader level of specificity (selection of
substrates is also based on its binding affinity to the receptor). GPCRs
have subtypes of receptors within
subfamilies with their own specificity which can be taken advantage of if
inhibiting a certain subtype is needed, yet a current problem in GPCR
agonists/antagonists is having low specificity. Targeting certain subtypes is
difficult since some drugs will readily bind to various receptor subtypes. In
contrast, the COX-1 and 2 receptors have a difference in substrate specificity
that has been used to develop specific drugs (such as celecoxib) for COX 2.
GPCRs such as the cholinergic receptors have a level of specificity employed for
the development of many quaternary ammonium muscle relaxants that mimic
acetylcholine's structure, but as discussed earlier, selectivity may not be
specific enough for the cholinergic subtypes. GPCR specificity is still not
fully elucidated and it may result from the cooperation of factors such as
interactions between the transmembrane helices, intracellular post-translation
modification, and dynamic structure of the extracellular active loop6, 7,
11. Determination of the interplay
between these factors should be sought to fully understand the degree of
specificity in GPCRs. In conclusion, there is a very high level of specificity
in COX receptors (a mere removal of a -CH2- group to reduce steric
collisions) in comparison to GPCRs (a broad group of proteins whose specificity
cannot be narrowed down to one amino acid substitution) which still require
further research to identify the hidden sources of receptor subtype specificity.
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