Molecular determinants in the carboxyl-terminus of a GPCR

The role of accessory proteins in G protein-mediated signaling is being increasingly appreciated. G proteins have long been considered the unique mediator in signal transduction by a G protein coupled 7-transmembrane receptor (GPCR) and it has become clear that most of the cytoplasmic peptide domains of the receptor contribute to the G protein interface. The peripheral carboxyl-terminal tail, however, appears to be dispensable for productive G protein coupling and is highly diverse in peptide sequence and length. The c-terminus likely presents a docking site for accessory non-G protein type proteins which interact with the receptor and an increasing number of instances can be given where this is the case. It is therefore sensible to assume that the c-terminus has evolved as an individualized domain with implications for signaling, sorting and regulation of and by the receptor. In the present application we propose to test this hypothesis using the A1-adenosine receptor as an prototypical model. Preliminary experiments revealed that the c-terminus serves two distinct roles. First, the membrane- proximal peptide stretch is required for receptor surface expression and for the ability of the receptor to bind ligand. Secondly, the peripheral c-tail reduces the affinity of the receptor for the cognate G protein thereby impeding activation of the signaling pathways. We have therefore performed a genebank interactor hunt and identified two candidate binding partners of the A1-adenosine receptor (the D1-dopamine receptor interacting protein, DRiP78 and astrin, a microtubulin binding protein). In the first part of the current application we propose to evaluate the role of one of the putative interaction partners (DriP78) in processing and export of the A1- adenosine receptor. By pursuing this approach we shall address if folding of the receptor is necessary and sufficient to pass the ER-Golgi quality control mechanism and if the interaction of the c-terminus with a chaperone is a critical step in receptor folding and export. In the second part, we propose to evaluate the role of the c-terminus in targeting of the A1-adenosine receptor to specific compartments in neuronal cells. In addition, we will determine if the c-terminus modulates signaling (signaling efficiency and selectivity for effectors) through association with accessory components (e.g. astrin).

The working hypothesis was that cell surface receptors belonging to the large class of G-protein-coupled receptors interact with accessory proteins that impinge on signalling, targeting and regulation by and of a given receptor. Single point mutations in the amino acid sequence of the A 1 adenosine receptor generated receptor versions that fail to be exported to the cell surface but are retained at the site of their biosynthesis, that is the endoplasmic reticulum. Hence, these receptor variants represent model molecules for the investigation of disorders linked to the aberrant retention of otherwise functional plasma proteins (e.g cystic fibrosis where the cystic fibrosis transmembrane regulator is blocked from export due to a discrete amino acid alteration). For the A 1 -receptor variants the deficiency can be remedied by treatment with cell-permeable receptor ligands ("pharmacochaperones") which help the receptor overcome the barrier at the exit gate of the endoplasmic reticulum. Because "pharmacochaperoning" is a potential application of drug therapy we have continued research to elucidate the specific mechanism by which ligands release the receptors from intracellular retention. Our most important findings were as follows: 1. The pharmacochaperone ligands bind to receptor folding intermediates in the endoplasmic reticulum. Ligand binding induces exiting of the receptor molecules from the endoplasmic reticulum followed by their transfer to the cell surface. All receptor variants then are fully functional. 2. The reason why the receptor variants are retained is unlikely due to a major folding problem; pharmaco- chaperones do not act as folding scaffolds. Hence in a bacterial organism that is devoid of an organelle as an endoplasmic reticulum and where receptors are inserted directly into the plasma membrane during biosynthesis of the peptide chain pharmaco-chaperones fail to increase the expression level. Rather, the effect of the pharmaco- chaperone is by reducing the retaining force of ancillary proteins which constitute the quality control machinery at the exit gate. 3. One of these proteins is DriP78 (the dopamine receptor interacting protein 78) which compared to the wild-type receptor preferentially recognizes the mutant A1 -receptor variants and the over-expression of which outdoes the action of the pharmacochaperones. Thus the pharmacological principle that may be derived from our experiments (performed with the support of FWF grant P16083) is that it is feasible to induce a fit in retained plasma proteins which results in the unlinking of associated proteins and thus in the enhanced export to the cell surface. Pharmacochaperoning may represent an indication in pharmacotherapy.