Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in postendocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the ␤ 2 -adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tailfusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G proteincoupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the ␦-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK 1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with Nethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrumrecycling adaptor.Interaction of receptors with adaptor and scaffolding proteins is important for their biogenesis, their cellular sorting and targeting to the cell membrane, and their function at the membrane in complex with transducer molecules and downstream effector molecules as well as the subsequent internalization and post-endocytotic sorting of the receptors (1, 2). These interactions among receptors, adaptors, and scaffolding proteins are highly regulated processes that can be controlled by phosphorylation events (3), expression of receptor activating, or inactivating variants of adaptor proteins (4), by competition among adaptor proteins, and by competition between adaptor proteins and effector molecules (5). For the large family of G protein-coupled seven-transmembrane segment receptors (7TM 1 receptors), this field is still in its infancy and only a rather sketchy picture has emerged of relative importance of specific adaptor and scaffolding proteins for the biogenesis, function, and desensitization of these receptors. Methods such as yeast two-hybrid screening...

The mechanisms underlying targeted sorting of endocytosed receptors for recycling to the plasma membrane or degradation in lysosomes are poorly understood. In this report, the C-terminal tails of the ¢ve dopamine receptors (D1^D5) were expressed as glutathione S-transferase (GST) fusion proteins and studied for their interaction with ezrin^radixin^moesin-binding phosphoprotein 50 (EBP50) and N-ethylmaleimide-sensitive factor (NSF), which are known to be involved in postendocytic recycling of receptors back to the plasma membrane, and with sorting nexin 1 (SNX1), known to be involved in targeting receptors to lysosomal degradation. EBP50 did not bind any of the dopamine receptor tails. NSF bound strongly to D1 and D5 and only weakly to D2, D3 and D4. However, SNX1 clearly distinguished between D1 and D5, as only D5 bound strongly to this protein. This report shows that there are distinct interaction patterns for NSF and SNX1 to the various dopamine receptor subtypes. ß