G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html.

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

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Dopamine D 4 receptors (D 4 Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D 4.2 R, D 4.4 R and D 4.7 R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo-and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D 4 R biogenesis and quality control in the endoplasmic reticulum plays an important role for D 4 R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D 4.4 R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D 4 R. The different polymorphic repeat variants of the D 4 R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo-and heterodimers. In summary, we conclude that D 4 Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.Structured digital abstract l D4.4R physically interacts with D4.2R by anti tag coimmunoprecipitation (View interaction) l D4.xR physically interacts with D4.4R by anti tag coimmunoprecipitation (View interaction) l D4.xR and D4.xR physically interact by bioluminescence resonance energy transfer (View interaction)Abbreviations BRET, bioluminescence resonance energy transfer; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino-2-phenylindole; D n R, dopamine D n receptor; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; MP, milk powder; YFP, yellow fluorescent protein.