Survey of Experimental Approaches and Representative Data

Flow Cytometric Approaches to Assess GPCR Function In Vivo 

A series of empirical schemes targeting the assembly of ternary complexes on beads have been attempted with varying degrees of success (reviewed in Simons et al, 2005). In Fig. 1, the molecular assemblies that are the subject of this chapter are based on the tethering of G protein heterotrimers to beads by an epitope tag (either hexahistidine or FLAGt ) fused to the N-terminus of the y subunit, and projected away from its binding partners in three-dimensional reconstructions. 

Detergent-solubilized receptors originate from cells that are transfected to overexpress the receptor of choice. The unpurified solubilized extracts contain other proteins including endogenous G proteins that are present in much higher concentrations than even the overexpressed target receptor. In normal cells, the expression of G proteins can be as much as 10-100 times higher than receptors (Ransnas and Insel, 1988). For the overexpressed receptor systems described here, it was determined by Western 

Molecular Assembly Data from Soluble Ligand, Ligand-Bead, and G-Bead Data 

Receptor Ligand Receptor affinity (fiM) EC50 on G-bead6 (/tM) Max receptor on G-beadc (No. per bead) Classification 4 

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