Soluble receptor ternary complex assemblies

III. Analysis of Soluble Receptor Ternary Complex Assemblies. 104... 

E. Ternary Complex Analysis of Soluble Receptor Assemblies. Ill... 

A measurement system that is able to quantitatively determine the interactions of receptor and G protein has the potential for more detailed testing of ternary complex models. The soluble receptor systems, ([l AR and FPR) described in Section II, allow for the direct and quantitative evaluation of receptor and G protein interactions (Simons et al, 2003, 2004). Soluble receptors allow access to both the extracellular ligandbinding site and the intracellular G protein-binding site of the receptor. As the site densities on the particles are typically lower than those that support rebinding (Goldstein et al, 1989), simple three-dimensional concentrations are appropriate for the components. Thus, by applying molar units for all the reaction components in the definitions listed in Fig. 2A, the units for the equilibrium dissociation constants are molar, not moles per square meter as for membrane-bound receptor interactions. These assemblies are also suitable for kinetic analysis of ternary complex disassembly. 

GPCR interactions with ligand and G protein are represented by the ternary complex formalism (Fig. 2A Christopoulos and Kenakin, 2002 De Lean et al, 1980 Sam am a et al, 1993). The quantitative analysis of the soluble assembly system formally requires inclusion of soluble G protein due to the use of a crude receptor preparation. These soluble G proteins compete with the G protein attached to the G-beads for the solubilized receptor as shown in Fig. 2C (Simons et al, 2003, 2004). Experimental values from G-beads (Fig. 3) were fitted with the calculations of bead-bound receptors (RG k..i< + ARG k. i< ) based on this model, which includes soluble G proteins. Simulations were made by Mathematica , numerically solving the series of... 

Analysis of the soluble G-bead assembly provides a complementary classification of full and partial agonists, based on their distinct abilities to assemble ternary complexes (LRG). It appears that the behavior of receptors and entire ligand families can be described by the simple ternary complex model alone (Fig. 2A). The analysis provides estimates for the ligand-dependent equilibrium constants that govern the simple ternary complex model. Unique, potentially intermediate, conformational states of the receptor defined by interactions with a particular ligand are characterized by individual binding constants. While these data do not directly show these different conformational states, the bead system appears to act as a... 

The LC then targets and cleaves one or more of three soluble NSF attachment protein receptor (SNARE) proteins with exquisite specificity synaptosomal-associated protein 25 (SNAP-25 BoNT/A, /C, /E) vesicle associated membrane protein 1-3 (VAMPl-3 BoNT-/B, /D, /F, /G) or syntaxin (BoNT/C). The SNARE proteins are essential components of the synaptic exocytosis mechanism, and their cleavage prevents functional assembly of the ternary complex, thereby blocking neurotransmitter release. The combination of efficient neuronal targeting and presynaptic activation renders BoNTs the most potent substances known, with estimated human lethal doses as low as 0.1-1 ng/kg. 

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