Membranes bound receptor modelling

There are no inherent limitations to the nature of the interaction that can be probed with the FAC method. This too stems from an uncoupling of the binding event and the detector. The method can be applied to simple binary interactions between protein and small molecule, but also to protein-protein interactions, protein-cell interactions and virtually any interaction that can be modeled in a flow system. Some of the more elegant examples include drug interaction with whole cells [12] and membrane-bound receptors from brain homogenates [13]. Ultimately, the limitations are dictated by what can be detected from a stream of column effluent. 

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. 

Initially, we attempted to build a 3-D homology model of the transmembrane regions of the human thrombin receptor from the structure of IBRD, which is available from the Brookhaven Protein Database. Unfortunately, the suboptimal placement of several amino acid side chains resulted in severe deviations from structural standards for membrane-bound receptors with a seven-helix bundle topology (Figure 2). For example, carboxylate and ammonium groups on amino acid side chains at a mid-helix location were directed into the membrane, rather than toward the inside of the helix bundle, and the hydrophobic packing between some helices was either poor or nonexistent. 

Genistein has been used for years as a model inhibitor of protein tyrosine kinases (PTKs). This ubiquitous enzyme, which is also present in thyroid foUicular ceUs, is involved in phosphorylation of tyrosyl residues of membrane-bound receptors leading to signal transduction. One of the signal transduction cascades leads to topoisomerase II, which participates in DNA replication. Blocking PTK activity is one of the mechanisms believed to be responsible for the anticancer potential of genistein. For reviews addressing this issue, which are beyond the scope of this chapter, see Peterson (1995), and Ravindranath et al (2004). 

Figure 9.3. Model for the action of humic substances (HS) on plasma membrane-bound targets of a root hair cell. Besides the well-known effects on plasma membrane H+-ATPase (P) and carriers (C) of mineral nutrients, the envisaged alteration of membrane lipid environment and the possible interaction with an hypothetical membrane receptor (R) for humic molecules which allows transduction of the signal for induction and expression of genes involved in nutrient uptake and root hair development are also presented.

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