Constitutive Activity of GPCRs and Pathophysiology of Disease

Constitutive activity of GPCRs may be of relevance for disease development and progression. Firstly, mutations in GPCR genes may cause disease by inducing constitutive activity. Secondly, constitutive activity of GPCRs may be relevant for infectious disease development, as several viruses contain constitutively active receptors in their genomes (see Chapter 15) 12 . Below, a few examples should illustrate how GPCR mutations and virally encoded GPCRs may affect human pathophysiology (Figure...

Analysis of Constitutive GPCR Activity in Membranes and Intact Cells

Studies with membranes have the advantage ofbeing able to eliminate contaminating agonists that may cause an apparent constitutive GPCR activation through multiple rounds of membrane centrifugation and resuspension (see also Chapter 1). In addition, studies with membranes allow for precise control ofthe concentrations of GTP and ions and also of pH, all of which have an effect on constitutive GPCR activity (see Chapter 9) 21-25 . Because ofthe striking sensitivity ofthe apparent constitutive...

Procedure for Sf9 Cell Culture and Membrane Preparation

Sf9 cells are cultured in 125-500 mL disposable Erlenmeyer flasks at 28 C with rotation at 150 rpm in SF 900 II medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5 (vol. vol.) fetal bovine serum (Bio Whittaker, Walkersville, MD, USA) and 0.1 mg mL-1 gentamicin (Roche, Indianapolis, IN, USA). Although fetal bovine serum is not absolutely necessary, Sf9 cells grow better and show higher GPCR expression levels in the presence of serum. Cells are maintained at a density of 1.0-6.0 x 106...

Kinetics of Agonist Binding and Receptor Activation

Recent studies with the b2AR 67-69 have challenged the existence of a preformed binding site for the agonist (i.e., the lock-and-key scheme where the agonist is simultaneously coordinated by the residues involved in binding the ligand). Instead, it has been suggested that the binding of the agonist and subsequent receptor activation follow a sequential process 4, 68-70 . The sequential binding model operates with several intermediate conformational states between the inactive and the fully...

Methodological Approaches

8.2 Analysis of Constitutive GPCR Activity in Membranes and Intact Cells 82 8.2.1 Procedure for Sf9 Cell Culture and Membrane Preparation 84 8.2.2 GPCR Radioligand Binding Studies 86 8.2.4 35S GTPyS Binding Assay 96 8.2.5 Adenylyl Cyclase Assay 101 8.3 Measurement of Constitutive Activity of GPCRs in Intact Cells 106 8.3.1 Quantitative Determination of cAMP Concentrations in Cell Culture Lysates 109 8.3.2 Determination of Inositol Phosphate Formation in Living Cells 110 8.3.3 Determination of G...

Physiological Relevance of Inverse Agonists

Several examples of endogenously expressed inverse agonists have been reported. The best characterized endogenous inverse agonist is retinal, which is bound to the light sensor rhodopsin 24 . Rhodopsin has evolved a unique mechanism to minimize basal receptor activity. The chromophore 11-cis-retinal, which acts as an inverse agonist in rhodopsin, is covalently bound to the receptor to ensure extremely low receptor signaling in the dark. Thus, 11-cis-retinal is responsible for the fact that the...

GPCR Radioligand Binding Studies

Good starting points for analysis of constitutive GPCR activity by radioligand binding studies are the availability of i an affordable and stable high-affinity tritiated radioligand, and ii various GPCR isoforms with different constitutive activities. The radioligand should preferably be a neutral antagonist, since the binding of neutral antagonists per se is not changed by G proteins 23, 32 , allowing for the analysis of the impact of G proteins on the binding of both inverse agonists and...