Early models for GPCR activation

Earlier mechanistic hypotheses largely emphasised the fluid-mosaic model of plasma membrane structure (Singer and Nicolson, 1972) and the notion of 'collision coupling' of the components. Implicit in this explanation was the idea that GPCRs, G proteins and effector components were physically separate entities that were free to diffuse in the membrane. Models that gathered widespread acceptance in the late seventies and eighties are the Collision Coupling Model (Tolkovsky and Levitzki, 1978) and the Ternary Complex Model for GPCR activation (De Lean et al., 1980) (Figure 156). Please note that, in this early models, R was inactive and that RG and ARG complexes were active (and represented the stimulus S); i.e. no attention was paid to the need of the receptor to adopt an active conformation before G protein coupling.

Figure 155 Pull-down assay to compare the relative affinities of different cytoplasmic proteins for the endo3 loop of a2A-adrenergic receptors (Wang and Limbird, 2002, reproduced by permission for the American Society for Biochemistry and Molecular Biology).
Figure 156 A: The Collision Coupling Model for GPCR activation by Tolkovski and Levitzki (Tolkovsky and Levitzki, 1978) and B: the Ternary Complex Model (De Lean et al., 1980, reproduced by permission for the American Society for Biochemistry and Molecular Biology).

Key features of this model are:

• GPCRs and effector components do not come into contact with one another but rather communicate via shuttling G proteins.

• Receptors have access to all cognate G proteins present at the cell surface. The rate of G protein activation is proportional to the collision frequency between the agonist-receptor complex (AR) and the trimeric G protein; i.e. it is proportional to [AR] and [G]. The formation of the ternary ARG complex is slow compared to G protein activation (i.e. the step with kact as rate constant). This means that the ARG complex is only transient in the presence of guanine nucleotides like GTP.

ARG complexes can be formed with agonists and these complexes display higher agonist affinity (i.e. KiH) as compared to AR (i.e. KiL). In fact, the presence of at least two distinct binding sites on the same receptor protein, one for the agonist and one for the G protein represents a simple example of an allosteric interaction: the agonist increases the receptor's affinity for the G protein and the G protein increases the affinity of the receptor for the agonist. G protein activation cannot take place in the absence of guanine nucleotides so that the ternary ARG complex is allowed to accumulate. High-affinity binding of radiolabelled agonists to cell membranes is therefore largely due to the formation of ternary ARG complexes.

In contrast, antagonists (I) will bind to the receptor without producing ternary IRG complexes. Hence, all receptors of the same kind will behave as a single class of antagonist binding sites. Agonist versus labelled antagonist competition binding curves on cell membrane preparations are often shallow in the absence of guanine nucleotides (Figure 157). This can be explained by:

• The high affinity component of the competition binding curve corresponds to ARG while the low affinity component corresponds to AR.

• Not all receptors can form an ARG complex at the same time. The original explanation (De Lean et al., 1980) was a stoichiometric limitation in the amount of available G proteins.

It seems that the KiL/KiH-ratio in agonist/labelled antagonist competition binding curves on membranes can be used to predict agonist intrinsic efficacy. When comparing such data with agonist concentration-response curves, please remember that the receptor reserve can mask partial agonist activity, and so result in a misleading classification of ligands.

When an excess of GTP (or analogues such as the non-hydrolyzable analogues Gpp(NH)p or GTPyS) is added, GTP versus GDP exchange can adequately take place so that the ARG complex falls apart in AR, GTP-bound-Ga and P-y This process

Agonist concentration (bars = Log intervals)

agonist competition binding (-GTP) A t R + G

agonist competition binding (+G TP) A +■ R + G short half-lire of ARB

compare to: antagonist competition binding (iGTPj I + R + G

Figure 157 Receptor-G protein coupling: effect of GTP on agonist versus radiolabelled antagonist competition binding, when GTP is present only low agonist affinity binding sites are detected.

is likely to proceed swiftly so that the concentration of ARG remains virtually nil (Figure 157). This adequately explains why:

• Binding of radiolabelled agonists to membrane preparations is greatly reduced in the presence of GTP.

• The shallow agonist competition binding curves become steep and only display the low affinity component in the presence of GTP.

High affinity agonist saturation and/or competition binding in transfected cell systems can only be expected when the appropriate G protein is present in sufficient quantities to produce observable ternary complexation.

The simplicity of the ternary complex model makes it easy to verify its validity. To deal with new experimental findings, the model has been adapted over the years. Major modifications deal with:

• Restricted GPCR-G protein coupling.

• Constitutive receptor activity.

low affinity high affinity

• Receptor coupling to different G proteins.

• Multiple affinity states of the receptor.

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