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C6. Agonist and Antagonist of Ligand Binding to Receptors - An Extension - Biology


The analysis of competitive, uncompetitive and noncompetitive inhibitors of enzymes can now be extended to understand how the activity of membrane receptors are affected by the binding of drugs. When receptors bind their natural target ligands (hormones, neurotransmitters), a biological effect is elicited. This usually involves a shape change in the receptor, a transmembrane protein, which activates intracellular activities. The bound receptor usually does not directly express biological activity, but initiates a cascade of events which leads to expression of intracellular activity. In some cases, however, the occupied receptor actually expresses biological activity itself. For example, the bound receptor can acquire enzymatic activity, or become an active ion channel.

Drugs targeted to membrane receptors can have a variety of effects. They may elicit the same biological effects as the natural ligand. If so, they are called agonists. Conversely they may inhibit the biological activity of the receptor. If so they called antagonists

Agonist

An agonist is a mimetic of the natural ligand and produces a similar biological effect as the natural ligand when it binds to the receptor. It binds at the same binding site, and leads, in the absence of the natural ligand, to either a full or partial response. In the latter case, it is called a partial agonist. The figure below shows the action of ligand, agonist, and partial agonist.

There is another kind of agonist, given the bizarre name inverse agonist. This term only makes sense when applied to a receptor that has a basal (or constitutive) activity in the absence of a bound ligand. If either the natural ligand or an agonist binds to the receptor site, the basal activity is increased. If however, an inverse agonists binds, the activity is decreased. An example of an inverse agonist (which we will discuss later) is the binding of the drug Ro15-4513 to the GABA receptor, which also binds benzodiazepines such as valium. When occupied by its natural ligand, GABA, the protein receptor is "activated" to become a channel allowing the inward flow of Cl- into a neural cell, inhibiting neuron activation. Valium potentiates the effect of GABA, which is enhanced even further in the presence of ethanol. Ro15-4513 binds to the benzodiazepine site, which leads to the opposite effect of valium, the inhibition of the receptor bound activity - a chloride channel.

Figure 1: Agonist and Partial Agonists

Antagonists

As there name implies, an antagonist inhibit the effects of the natural ligand (hormone, neurotransmitter), agonist, partial agonist, and even inverse agonists (which will not be mentioned again). We can think of them as inhibitors of receptor activity, much as we considered in the sections above inhibitors of enzyme activity. As such, there can be different types of antagonists. These include:

  • Competitive antagonist, which are drugs that bind to the same site as the natural ligand, agonists, or partial agonist, and inhibit their effects. They would be analogous to competitive inhibitors of enzyme. One could also imagine a scenario in which an "allosteric" antagonistbinds to an allosteric site on the receptor, inducing a conformational change in the receptor so the ligand, agonist, or partial agonist could not bind.
  • Noncompetitive antagonist (or perhaps more generally mixed antagonist) which are drugs that bind to a different site on the receptor than the natural ligand, agonist, or partial agonist, and inhibit the biological function of the receptor.In analogy to noncompetitve and mixed enzyme inhibitors, the noncompetitive antagonist may change the apparent (K_d) for the ligand, agonist, or partial agonist (the ligand concentration required to achieve half-maximal biological effects), but will change the maximal response to the ligand (as mixed inhibitors change the apparent (V_{max}). The figure below shows the action of a competitive and noncompetitive antagonist.
  • Irreversible agonist, which arises from covalent modification of the receptor.

Figure 2: Antagonists: Competitive and Noncompetitive (Mixed)


C6. Agonist and Antagonist of Ligand Binding to Receptors - An Extension

The analysis of competitive, uncompetitive and noncompetitive inhibitors of enzymes can now be extended to understand how the activity of membrane receptors are affected by the binding of drugs. When receptors bind their natural target ligands (hormones, neurotransmitters), a biological effect is elicited. This usually involves a shape change in the receptor, a transmembrane protein, which activates intracellular activities. The bound receptor usually does not directly express biological activity, but initiates a cascade of events which leads to expression of intracellular activity. In some cases, however, the occupied receptor actually expresses biological activity itself. For example, the bound receptor can acquire enzymatic activity, or become an active ion channel.

Drugs targeted to membrane receptors can have a variety of effects. They may elicit the same biological effects as the natural ligand. If so, they are called agonists. Conversely they may inhibit the biological activity of the receptor. If so they called antagonists

An agonist is a mimetic of the natural ligand and produces a similar biological effect as the natural ligand when it binds to the receptor. It binds at the same binding site, and leads, in the absence of the natural ligand, to either a full or partial response. In the latter case, it is called a partial agonist. The figure below shows the action of ligand, agonist, and partial agonist.

There is another kind of agonist, given the bizarre name inverse agonist. This term only makes sense when applied to a receptor that has a basal (or constitutive) activity in the absence of a bound ligand. If either the natural ligand or an agonist binds to the receptor site, the basal activity is increased. If however, an inverse agonists binds, the activity is decreased. An example of an inverse agonist (which we will discuss later) is the binding of the drug Ro15-4513 to the GABA receptor, which also binds benzodiazepines such as valium. When occupied by its natural ligand, GABA, the protein receptor is "activated" to become a channel allowing the inward flow of Cl- into a neural cell, inhibiting neuron activation. Valium potentiates the effect of GABA, which is enhanced even further in the presence of ethanol. Ro15-4513 binds to the benzodiazepine site, which leads to the opposite effect of valium, the inhibition of the receptor bound activity - a chloride channel.

As there name implies, antagonist inhibit the effects of the natural ligand (hormone, neurotransmitter), agonist, partial agonist, and even inverse agonists. We can think of them as inhibitors of receptor activity, much as we considered in the sections above inhibitors of enzyme activity. As such, there can be different types of antagonists. These include:

  • competitive antagonist, which are drugs that bind to the same site as the natural ligand, agonists, or partial agonist, and inhibit the effect of the natural ligand or agonist. They would be analogous to competitive inhibitors of enzyme. One could also imagine a scenario in which an "allosteric" antagonist binds to an allosteric site on the receptor, inducing a conformational change in the receptor so the ligand, agonist, or partial agonist could not bind.
  • noncompetitive antagonist (or perhaps more generally mixed antagonist) which are drugs that bind to a different site on the receptor than the natural ligand, agonist, or partial agonist, and inhibit the biological effect of the natural ligand or agonist. In analogy to noncompetitve and mixed enzyme inhibitors, the noncompetitive antagonist may change the apparent KD for the ligand, agonist, or partial agonist (the ligand concentration required to achieve half-maximal biological effects), but will change the maximal response to the ligand (as mixed inhibitors change the apparent Vmax. The figure below shows the action of a competitive and noncompetitive antagonist.
  • irreversible agonist, which arises from covalent modification of the receptor.

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Agonist drugs

The International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (Neubig et al, 2003) give an "official" definition as

"Agonist: A ligand that binds to a receptor and alters the receptor state resulting in a biological response"

Which sounds a lot like the definition of drug effect (as in effect that's what it is). There is a number of different definitions, and generally they are all either an echo of the statement above, or an attempt to explain the concept with an example. It would be unexpected to encounter the need to define "agonist" in an exam scenario, as the meaning of the term is intuitively understood by most, and the task of defining it is more of a test of English language control, which probably does not belong in a higher-order medical college examination. One would be more likely to be asked to describe what the differences are between different types of agonists.


Agonists

Agonists are both endogenous and exogenous – these are small molecules which form an agonist receptor complex resulting in the activation of the receptor. Agonists are a type of ligand.

Agonists usually bond to a receptor through reversible chemical bonding. This requires bonding between the agonist and the ligand. Usually a long range force in the form of electrostatic bonding will ‘draw’ the agonist and receptor together. Following this hydrogen bonding and Van der walls bonding will allow for a close bonding. Sometimes hydrophobic bonding will also occur, however, this is not always the case.

Recall the concept of equilibrium where the rate of forward reaction is equal to the rate of reverse reaction. For reversible binding of ligands to receptors, this also holds true.

[(A) Drug] + [(R) vacant receptor] [(AR) drug-receptor complex]

The forward reaction is k+1 and the reverse reaction is k-1

A hydrogen isotope attached to the ligand is typically used in experiments to measure this, this will release radiation, allowing us to pinpoint where it is.

Therefore, the kd = k-1/k+1 this has units of moles/L. This is where half the receptors are occupied by the agonist.

The affinity is thus k+1/k-1 which is the reciprocal of the kd, therefore, an increase in the affinity results in more drug-receptor complex formation.

The kd can be used to determine what concentration of drug is effective for desired response in vivo (in living system). This is often the concentration at 3 times the kd.

https://www.sciencedirect.com/science/article/pii/S1319016416300706

If with graph the concentration of drug against the pharmaceutical effect we get a rectangular hyperbole. If we log the concentration of drug we then get a sigmoidal curve.

This can be represented by the equation:

The specific binding is found to be the total bound drug – non specific binding. This is how we find the direct binding.

Direct binding can then be sketched using a scatchard plot – this can be used to find the KD of the drug.

This is the [bound drug]/[[free drug] on the y axis against [bound] which gives a linear straight line. The x intercept is the B max where the larger the number, the more receptors. The gradient is the negative inverse of the KD.

The concentration response curves, as seen above, can also be represented with the equation:

response/ maximum response = XA / (XA + EC50)

Emax or Bmax is the total physiological response.

This is derived from the functionality assay.

https://en.wikipedia.org/wiki/Hill_equation_(biochemistry)

When the hill coefficient (α) is:

  • greater then 1= positive cooperativity in the receptor e.g hemoglobin (2.8-3)
  • less then 1= negative cooperativity in the receptor (partial agonist) e.g insulin (.7-.8)
  • 0 = antagonist binding

We can now start to compare different agonists.

We can use the maximum response which is the drugs intrinsic activity divided by its effectivness.

  • Maximal physiological response is a measure of drug efficacy
  • EC50 (likewise pD2) is used for potency, where it is half the concentration of a maximum response)
  • ED50 is half the dosage for a maximum response
  • Intrinsic activity is interchangeable most of the time is efficacy which provides the maximum response.

Bolus concentration response

The drug is administered, and response is measured then the drug is washed out and and tissue recovers. This is then repeated and the concentration of drug administered is increased.

Cumulative concentration response

Add drug and measure response, add another dose before it recovers, increasing at fixed intervals. Once the response plateaus out – the Bmax is found.

Explain why a drug being prescribed close to it’s EC50 will have a significant increase in its response for a biological system if it’s dosage is doubled, however, this increase in response is less significant when a drug being prescribed significantly greater then it’s EC50 is prescribed.


Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT1B receptors in C6-glial cells: similarities with the human 5-HT1B receptor

The guinea-pig recombinant 5-hydroxytryptamine1B (gp 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by monitoring G-protein activation in a membrane preparation with agonist-stimulated [ 35 S]-GTPγS binding. The intrinsic activity of 5-HT receptor ligands was compared with that determined previously at the human recombinant 5-HT1B (h 5-HT1B) receptor under similar experimental conditions.

Membrane preparations of C6-glial/gp 5-HT1B cells exhibited [ 3 H]-5-carboxamidotryptamine (5-CT) and [ 3 H] - N- [4-methoxy-3,4 - methylpiperazin-1-yl) phenyl] -3 - methyl -𠂔-(4 - pyridinyl)benzamide (GR 125743) binding sites with a pKd of 9.62 to 9.85 and a Bmax between 2.1 to 6.4𠂟mol mg 𢄡 protein. The binding affinities of a series of 5-HT receptor ligands determined with [ 3 H]-5-CT and [ 3 H]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r 2 : 0.74, Pπ.001) with those determined at the recombinant h 5-HT1B receptor.

[ 35 S]-GTPγS binding to membrane preparations of C6-glial/gp 5-HT1B cells was stimulated by the 5-HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitriptan, sumatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethylsulphonamide (CP122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5-HT. The potency of these agonists showed a positive correlation (r 2 : 0.72, P=0.015) with their potency at the recombinant h 5-HT 1B receptor. 1-naphthylpiperazine, (±)-cyanopindolol and (2′-methyl-4′-(5-methyl[1,2,4] oxadiazole-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935) elicited an even smaller response (Emax: 0.32 to 0.63).

The ligands 1′-methyl-5-(2′-methyl-4′-(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro [furo[2,3-f]indole-3-spiro-4′-piperidine] (SB224289), methiothepin and ritanserin displayed inhibition of basal [ 35 S]-GTPγS binding at concentrations relevant to their binding affinity for the gp 5-HT1B receptor. Methiothepin and SB224289 behaved as competitive antagonists at gp 5-HT1B receptors pA2 values were 9.74 and 8.73, respectively when 5-HT was used as an agonist. These estimates accorded with the potencies measured in antagonism of zolmitriptan-mediated inhibition of forskolin-stimulated cyclic AMP formation. Ketanserin acted as a weak antagonist (pKB: 5.87) at gp 5-HT1B receptors.

In conclusion, the recombinant gp 5-HT1B receptor shares important pharmacological similarities with the recombinant h 5-HT1B receptor. The finding that negative activity occurs at these receptors further suggests that SB224289, methiothepin and ritanserin are likely to be inverse agonists.


Agonist vs antagonist medical definition

Drugs that stimulate the sympathetic nervous system are called adrenergic agonists, adrenergics, or sympathomimetics because they mimic the sympathetic neurotransmitters norepinephrine and epinephrine. A receptor antagonist is a type of receptor ligand or drug that blocks or dampens a biological response by binding to and blocking a receptor rather than activating it like an agonist. 5. This will permanently modify the receptor preventing the binding of the ligand. The antonym of antagonist is agonist. Antagonists and agonists are key players in the chemistry of … ersetzt. Increasing the ligand concentration can suppress the effect of the competitive antagonist. With a direct-acting antagonist, the drug works by taking up the space in neurotransmitters and receptors that would normally be filled by other transmissions. These drugs actually work in counteractive directions. Read medical definition of Beta-agonist. These are classified separately based on how they interact with neurotransmitters in the mind. The response is prevented when the antagonist binds to the binding site. In pharmacology the term agonist-antagonist or mixed agonist/antagonist is used to refer to a drug which under some conditions behaves as an agonist (a substance that fully activates the receptor that it binds to) while under other conditions, behaves as an antagonist (a substance that binds to a receptor but does not activate and can block the activity of other agonists). Agonist: A substance that acts like another substance and therefore stimulates an action. B. ein Hormon oder ein Neurotransmitter) als auch ein nicht-körpereigener Wirkstoff, der einen bestimmten Botenstoff in seiner Wirkung imitiert bzw. The mechanism of opioids can be explained by two mechanisms – the agonistic mechanism and the antagonistic mechanism. B. Click here for instructions on how to enable JavaScript in your browser. This is due to the shape of the antagonist which mimics the natural ligand. Inverse agonists – the simplest definition is that the com- pound binds to a receptor but produces the opposite effect from an accepted agonist. Um eine Bewegung ausführen zu können, ist immer das Zusammenspiel gegensätzlich wirkender Muskeln notwendig. Instead, they operate by blocking the brain’s neurotransmitters from functioning correctly. It has this name because it competes against neurotransmitters by blocking them from working. It may cause a change in the chemicals or reactions that take place within the brain by imitating the way a neurotransmitter normally operates. Most drugs operate in a variety of ways within the human body. This type of drug is designed to stimulate an action and can work to relax the muscles. Agonist is the opposite of antagonist. The Difference between Opiate Agonist and Antagonist … Agonists at this receptor enhance γ-aminobutyric acid (GABA) transmission, inverse agonists reduce GABA transmission and antagonists … Als Agonist (von altgriechisch αγωνιστής agonistēs der Tätige, Handelnde, Führende) wird in der Pharmakologie eine Substanz (Ligand) bezeichnet, die durch Besetzung eines Rezeptors die Signaltransduktion in der zugehörigen Zelle aktiviert. Thus the binding of the agonist drug results in similar biological effect as the natural ligand. One could also imagine a scenario in which an "allosteric" antagonistbinds to an allosteric site on the receptor, inducing a conformational change in the receptor so the ligand, agonist … A simple way to think about these concepts is that agonist therapy creates an action while antagonist therapy opposes an action. These drugs give delayed responses. They would be analogous to competitive inhibitors of enzyme. One of the most common types of indirect-acting agonist is the illicit drug, cocaine. Medical Definition of agonist. An agonist can be either a direct-binding agonist or and indirect-acting agonist. Agonist Therapy vs Antagonist Therapy. The efficacy of a full agonist is by definition 100%, a neutral antagonist has 0% efficacy, and an inverse agonist has


4 THE FUTURE OF RECEPTOR THEORY: KINETICS

One area of receptor theory that has tended to lag behind other disciplines within experimental pharmacology is that of system kinetics. Indeed, the time course of drug absorption, distribution, metabolism and elimination comprises the entire field of pharmacokinetics (reviewed in Fan & de Lannoy, 2014 Wright, Winter, & Duffull, 2011 )—yet the most widely utilised models in receptor theory remain fundamentally equilibrium based, including all of the EMAX model, two-state and other multistate models, ternary complex models and the operational model.

The incongruence of the equilibrium assumption with the nature of GPCR activity has been identified by some researchers to date. A notable example of this includes a study examining biased agonism at D2 dopamine receptors. In order to more closely examine the impact of receptor activity kinetics, operational analysis was performed at repeated intervals over an extended time course of assay detection in several pathways (Klein Herenbrink et al., 2016 ). Interestingly, both agonist potency rank order and bias conclusions for the ligands changed (sometimes even reversing) time-dependently for the pathways included. This was put down to different agonist association/dissociation kinetics—agonists that dissociated slowly from their receptors were more active at later time points (relative to other ligands) and fast-dissociating agonists were similarly favoured at earlier time points (Klein Herenbrink et al., 2016 ). Despite such approaches having useful diagnostic utility, this approach is undermined by the fact that operational analysis remains an equilibrium-based model (and therefore this “cross-sectional” analysis approach is somewhat simplistic) and hence does not provide evidence to quantify the magnitude of agonists' biases. True kinetic models are therefore required.

As derived above, a ligand's equilibrium dissociation constant, KD , is also defined as a ratio of two kinetic parameters, the dissociation and association rate constants ( ). Clearly, it is possible for different ligands to differ hugely in these parameters, which can be reflected in different signalling time courses even if their equilibrium dissociation constants are identical. Recent reports have started to examine ways to directly estimate these rate constants, to help explain different signalling pathway kinetics (reviewed in Sykes, Stoddart, Kilpatrick, & Hill, 2019 ). The intrinsically differing kinetics of different activity pathways will interact and confound the overall response time course. However, to date, very few models exist that directly and specifically account for both different ligand binding kinetics and different signalling pathway kinetics. Such an integrated model framework of receptor activity dynamics will be required, in order to explain all components (particularly different effect/activity time courses) of drug responses. We and others are pursuing research in this area currently (e.g. Bridge, Mead, Frattini, Winfield, & Ladds, 2018 Hoare, Pierre, Moya, & Larson, 2018 Zhu, Finlay, Glass, & Duffull, 2019 ). Further complexity originates from the contribution of receptor desensitisation and internalisation—these are themselves time- and agonist-dependent effects (Zhu et al., 2019 ) but inherently assert a type of “negative feedback” effect on the activity of other pathways. This type of regulatory effect has received little attention in GPCR functional modelling to date.

Given the hitherto pre-eminence of equilibrium models of receptor function, it may be surprising that thoughts about the kinetics of receptor pharmacology are not entirely new. Once again, AV Hill was the first to put forward a theoretical justification for non-instantaneous equilibrium in his work on the nicotine concentration–effect relationship in muscle (Hill, 1909 ). In this work, Hill derived λ , a parameter that is independent of the nicotine concentration. This new parameter implicitly explains an observed delay in nicotine-induced contractile responses between two different response compartments (intra- and extra-muscular) and thus added a kinetic dimension to Hill's otherwise equilibrium-based concentration–effect model (Hill, 1909 ). These concepts were discovered again in the 1970s and incorporated into the theory of in vivo drug concentration kinetics—the so-called biophase delay or hypothetical effect compartment (Sheiner, Stanski, Vozeh, Miller, & Ham, 1979 reviewed in Wright et al., 2011 ).

Progress in the area of receptor theory now depends on close collaboration between empirical pharmacologists and pharmacometricians, the former to produce data that can inform model development and inference by the latter and in turn the latter to produce utilitarian tools that can be applied by the former. It seems likely that the reason that the method for analysing functional selectivity using the operational model (Van Der Westhuizen et al., 2014 ) has become so widespread is that it was published as a drop-in method of analysis in GraphPad Prism. As research proceeds, and as more of the elements of receptor function are incorporated quantitatively into complex mechanistic models, it is to be hoped the “two-pronged,” co-dependent evolution of this important subject can be as productive in the future as it has been in the past.


Structural Diversity of the Adenosine Receptor Family

The amino acid sequence of the four AR subtypes is relatively poorly conserved, A2AR shares only 49, 56, and 39% identity with A1R, A2BR, and A3R, respectively (aligned over residues 1-312 of A2AR). This means that, despite there being a wealth of structural data available for A2AR, it has proved challenging to homology model other AR subtypes with sufficient accuracy for structure-based drug design applications (Glukhova et al., 2017). It is only during the past year that structures of an AR other than A2AR have been published, namely two structures of A1R bound to the xanthine antagonists DU172 (Glukhova et al., 2017) and PSB36 (Cheng et al., 2017). The two A1R structures are closely related and align with an RMSD of 0.6 Å (over 235 Cα atoms), they also align well with the ZM241385-bound structure of A2AR (Liu et al., 2012), with RMSDs of 0.8 Å (over 238 Cα atoms) and 1.0 Å (over 234 Cα atoms) for the DU172- and PSB36- bound structures, respectively. The intracellular side of both A1R structures strongly resemble the ZM241385-bound A2AR structure (3PWH), which was crystallized without a fusion protein in ICL3 (Doré et al., 2011), and the ionic lock between residues R105 3.50 and E229 6.30 is engaged in both cases (Cheng et al., 2017 Glukhova et al., 2017). The organization of the sodium-binding site is also well-conserved between A1R and A2AR, which supports mutagenesis data that indicated the negative allosteric effect of sodium on A1R was mediated through this site (Barbhaiya et al., 1996), although neither A1R structure was of sufficient resolution to conclusively model the sodium ion.

The most striking differences between A1R and A2AR are the conformational variations in extracellular ends of H1, H2, H3, and H7 and the orientation of ECL2. In the DU172-bound structure H3 is displaced inwards by 4 Å, and H1, H2, and H7 are displaced outwards by 5, 4, and 4 Å, respectively (Figure 6A). The outward movements in H1, H2, and H7 are required to accommodate the benzene sulfonate group of DU172, which is covalently linked to Y271 7.36 , and result in both the expansion of the orthosteric site and the formation of a secondary allosteric pocket (Glukhova et al., 2017). Direct comparison of A1R and A2AR bound to PSB36, which does not contain the benzene sulfonate substituent, also reveals a partial expansion of the orthosteric pocket (Cheng et al., 2017), indicating that this region of A1R is indeed more conformationally malleable than that of A2AR. Intriguingly, it has been suggested that the conformational rearrangements in H1, H2, and H3 in A1R may be a direct result of the different disulphide bond structure of ECL2 (Glukhova et al., 2017). In both A1R structures ECL2 adopts a similar conformation, which is different from that observed in any of the published A2AR structures (Figure 6B). The helical segment in ECL2 of A1R is extended by five residues and is positioned almost perpendicular to the transmembrane helices, compared to the near parallel arrangement in A2AR (Cheng et al., 2017 Glukhova et al., 2017). There is only a single disulphide bond in ECL2 of A1R compared to three within the same region of A2AR, which results in it adopting an extended conformation (Figure 6B). This appears to reduce conformational constraints on the extracellular ends of H2 and H3 allowing them to be displaced outwards, which in turn influences the positioning of H1. The displacement of H7 may also be linked to structural divergence in the extracellular loops, in this case a single amino acid truncation in ECL3 of A1R has been proposed to induce the outward tilt observed in the structures (Cheng et al., 2017 Glukhova et al., 2017).

Figure 6. Structural diversity of the adenosine receptor family. (A) Extracellular view of the conformational differences between DU172-bound A1R (colored magenta PDB: 5UEN) (Glukhova et al., 2017) and ZM241385-bound A2AR (colored cyan PDB: 4EIY) (Liu et al., 2012). The extracellular ends of H1, H2 and H7 are displaced outwards by 5, 4, and 4 Å, respectively and H3 is displaced inwards by 4 Å (indicated by red arrows). The antagonist DU172 (shown as spheres) is covalently attached to Y271 7.36 (shown as sticks) through a benzene sulfonate linkage. Note that for clarity, ECL2 has been omitted from the alignment. (B) Differential conformations of ECL1 (colored green) and ECL2 (colored blue) in A1R (colored magenta PDB: 5UEN) (Glukhova et al., 2017) and A2AR (colored cyan PDB: 4EIY) (Liu et al., 2012), disulphide bonds are shown as sticks. In A1R ECL2 adopts an extended conformation that is stabilized by a single disulphide bond (C80 3.25 -C169 ECL2 ). In contrast, ECL2 from A2AR adopts a compact conformation that is stabilized by three disulphide bond, one of which (C77 3.25 -C166 ECL2 ) is conserved in A1R. (C) Surface representation of the ligand-binding pocket (rendered as semi-transparent mesh) in A1R (colored blue PDB: 5N2R) (Cheng et al., 2017) and A2AR (colored green PDB: 5N2S) (Cheng et al., 2017), which highlights differences in both the topology of the orthosteric site and the binding orientation of PSB36 (shown as sticks). The butyl substituent at position N1 of the xanthine core of PSB36 fits into a channel between H3, H5, and H6 in A1R, this channel is constricted in A2AR due to the different orientation of L85 3.34 (shown as sticks), which results in the ligand binding in a different orientation.

What do these structures tell us about the molecular determinants of ligand-binding specificity in different AR subtypes? First, sequence differences in the binding pocket do not appear to be the main determinant of ligand-binding specificity in ARs. The orthosteric binding pocket of A1R and A2AR in the PSB36-bound structures differ by only four residues V62 2.57 , N70 2.65 , E170 ECL2 , and T270 7.35 (corresponding to A59 2.57 , S67 2.65 , L170 ECL2 , and M270 7.35 in A2AR), and of these, only T/M270 7.35 form direct contacts with the ligand (Cheng et al., 2017). Mutagenesis studies have shown that residue 270 7.35 is important in ligand-binding specificity, introducing the T270M mutation into A1R resulted in decreased binding affinity of A1R-specific ligands and increased binding affinity of A2AR-specific ligands, the reverse effect was observed when the M270T mutation was introduced into A2AR (Cheng et al., 2017 Glukhova et al., 2017). However, the positioning of this residue on the extracellular end of H7, at the perimeter of the binding pocket, suggests its main role is to act as a “gatekeeper” that regulates ligand access to the orthosteric site (Glukhova et al., 2017). Second, the topology of the binding pocket appears to play a central role in ligand-binding specificity. As described above the disulphide bond structure of ECL1 and ECL2 in A1R increases mobility in H1, H2 and H3, which causes an expansion of the binding pocket that is required to accommodate the benzene sulfonate group of the A1R-selective ligand DU172 (Figure 6A) (Glukhova et al., 2017). Binding pocket topology is also the predominant factor in the differential binding modes of PSB36 between A1R and A2AR (Figure 6C). PSB36 binds 2 Å deeper in the orthosteric pocket of A1R due to the presence of a channel between H3, H5 and H6 that can accommodate the butyl substituent at position N1 of the xanthine core (Figure 6C) (Cheng et al., 2017). This channel is created by a 2 Å displacement of L88 3.34 in A1R that appears to be the result of an upstream proline residue (P86 3.31 ), which is located outside the binding pocket, distorting the helix geometry in this region of H3. A proline at this position is unique to A1R and helps to explain why substitutions at position N1 of the xanthine core contribute to A1R selectivity (Cheng et al., 2017). Thus, the amino acid sequence both inside and outside the ligand-binding site, the extracellular loop structure and the topology of the binding pocket play interconnected roles in governing the ligand binding affinity and kinetics that are ultimately responsible for the functional selectivity of AR subtypes.


Acknowledgements

Research support from the National Institutes of Health (PHS 5R37DK015556 to J.A.K. 5R33CA132022, 5R01DK077085 to K.W.N. 1U01GM102148 to K.W.N and P.R.G., and 5R01CA130932 to J.F.), The Breast Cancer Research Foundation (to B.S.K.), BallenIsles Men's Golf Association (to J.C.N.), Frenchman's Creek Women for Cancer Research (to S.S.), Susan G. Komen for the Cure (PDF12229484 to IK), and the National Natural Science Foundation of China (81172935, 81373255, 81573279), Hubei Province's Outstanding Medical Academic Leader Program (to H.-B.Z.). Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or the NIH.


Abstract

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1−21) and Ucn1(32−40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(α) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2−C6), the disulfide arrangement C2−C5 and C4−C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11−40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry43, 3996−4011], a third binding domain should exist, probably comprising residues 8−10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.

This project was supported by the Deutsche Forschungsgemeinschaft (Grant SFB 449).

Research Institute of Molecular Pharmacology.

Martin-Luther University Halle-Wittenberg.

To whom correspondence should be addressed. Tel: +49-341-9715898. Fax: +49-341-9715949. E-mail: [email protected]


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