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Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development. [ABSTRACT FROM AUTHOR]

The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Truncated or shorter forms of G protein-coupled receptors (GPCRs), originating by alternative splicing, have been considered physiologically irrelevant for a rather long time. Nevertheless, it is now recognized that alternative splicing variants of GPCRs greatly increase the total number of receptor isoforms and can regulate receptor trafficking and signalling. Furthermore, dimerization of these truncated variants with other receptors concurs to expand receptor diversity. Highly truncated variants of GPCRs, typically, are retained in the endoplasmic reticulum (ER) and by heteromerization prevent the wild-type receptor to reach the plasma membrane, exerting a dominant-negative effect on its function. This can be responsible for some pathological conditions but in some other cases, it can offer protection from a disease because the expression of the receptor, that is necessary for binding an infectious agent, is attenuated. Here, we propose a possible new mechanism of creation of truncated GPCR variants through an internal ribosome entry site (IRES), a nucleotide sequence that allows cap independent translation of proteins by recruiting the ribosome in proximity of an internal initiation codon. We suggest that an IRES, situated in the third cytoplasmic loop, could be responsible for the translation of the last two transmembrane (TM) regions of the muscarinic M2 receptor. IRES driven expression of this C-terminal part of the muscarinic M2 receptor could represent a novel and additional mechanism of receptor regulation. [ABSTRACT FROM AUTHOR]

The introduction of super-resolution fluorescence microscopy has allowed the visualization of single proteins in their biological environment. Recently, these techniques have been applied to determine the organization of class A G-protein-coupled receptors (GPCRs), and to determine whether they exist as monomers, dimers and/or higher-order oligomers. On this subject, this review highlights recent evidence from photoactivated localization microscopy (PALM), which allows the visualization of single molecules in dense samples, and single-molecule tracking (SMT), which determines how GPCRs move and interact in living cells in the presence of different ligands. PALM has demonstrated that GPCR oligomerization depends on the receptor subtype, the cell type, the actin cytoskeleton, and other proteins. Conversely, SMT has revealed the transient dynamics of dimer formation, whereby receptors show a monomer-dimer equilibrium characterized by rapid association and dissociation. At steady state, depending on the subtype, approximately 30-50% of receptors are part of dimeric complexes. Notably, the existence of many GPCR dimers/oligomers is also supported by well-known techniques, such as resonance energy transfer methodologies, and by approaches that exploit fluorescence fluctuations, such as fluorescence correlation spectroscopy (FCS). Future research using single-molecule methods will deepen our knowledge related to the function and druggability of homo-oligomers and hetero-oligomers. [ABSTRACT FROM AUTHOR]

The brain׳s complexity derives not only from the way the intricate network of neurons is wired, but also by protein complexes that recognize and decode chemical information. G protein-coupled receptors (GPCRs) represent the most abundant family of proteins mediating neurotransmission in the brain, and their ability to form homo- and heteromers which amplifies the scope for synaptic communication and fine-tuning. Dopamine receptors are important drug targets and members of both the D 1 /D 5 and D 2 /D 3 /D 4 receptor families form homo- and heteromers. The present article focuses on D 3 receptor homo- and heteromers, in particular, those formed in association with their D 2 counterparts. We highlight the binding profiles and mechanisms of interaction with D 3 –D 3 homomers and D 3 –D 2 heteromers of: first, the PET ligand and potent agonist [ 11 C]-(+)-PHNO; second, the novel, bitopic/allosteric dopamine D 3 receptor antagonist, SB269,652; and third, diverse partial agonists like antipsychotic and aripiprazole. Molecular mechanisms of interplay between the two protomers of heteromeric D 3 –D 2 complexes are likewise discussed: for example, “transactivation”, whereby recruitment of one member of a heteromer harnesses signalling pathways is normally coupled to the other protomer. Finally, D 1 receptor heteromers are also taken into consideration in deciphering the nature of interfaces required to stabilize dimeric assemblies and permit their interaction with G proteins. Improved understanding of D 3 as well as D 2 and D 1 receptor complexes should yield important insights into their physiological roles and pathological significance, and permit the development of novel drug classes with potentially distinctive functional profiles and improved therapeutic windows. [ABSTRACT FROM AUTHOR]

"Ecstasy" [3,4-methylenedioxymetamphetamine (MDMA)] is of considerable interest in light of its prosocial properties and risks associated with widespread recreational use. Recently, it was found to bind trace amine-1 receptors (TA1Rs), which modulate dopaminergic transmission. Accordingly, using mice genetically deprived of TA1R (TA1-KO), we explored their significance to the actions of MDMA, which robustly activated human adenylyl cyclase-coupled TA1R transfected into HeLa cells. In wild-type (WT) mice, MDMA elicited a time-, dose-, and ambient temperature-dependent hypothermia and hyperthermia, whereas TA1-KO mice displayed hyperthermia only. MDMA-induced increases in dialysate levels of dopamine (DA) in dorsal striatum were amplified in TA1-KO mice, despite identical levels of MDMA itself. A similar facilitation of the influence of MDMA upon dopaminergic transmission was acquired in frontal cortex and nucleus accumbens, and induction of locomotion by MDMA was haloperidol-reversibly potentiated in TA1-KO versus WT mice. Conversely, genetic deletion of TA1R did not affect increases in DA levels evoked by para-chloroamphetamine (PCA), which was inactive at hTA1 sites. TheTA1Ragonist o-phenyl-3-iodotyramine (o-PIT) blunted the DA-releasing actions ofPCAboth in vivo (dialysis) and in vitro (synaptosomes) in WT but not TA1-KO animals. MDMA-elicited increases in dialysis levels of serotonin (5-HT) were likewise greater in TA1-KO versus WT mice, and 5-HT-releasing actions of PCA were blunted in vivo and in vitro by o-PIT in WT mice only. In conclusion, TA1Rs exert an inhibitory influence on both dopaminergic and serotonergic transmission, and MDMA auto-inhibits its neurochemical and functional actions by recruitment of TA1R. These observations have important implications for the effects of MDMA in humans. [ABSTRACT FROM AUTHOR]

Information flow from a source to a receiver becomes informative when the recipient can process the signal into a meaningful form. Information exchange and interpretation is essential in biology and understanding how cells integrate signals from a variety of information-coding molecules into complex orchestrated responses is a major challenge for modern cell biology. In complex organisms, cell to cell communication occurs mostly through neurotransmitters and hormones, and receptors are responsible for signal recognition at the membrane level and information transduction inside the cell. The G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, with nearly 800 genes coding for these proteins. The recognition that GPCRs may physically interact with each other has led to the hypothesis that their dimeric state can provide the framework for temporal coincidence in signaling pathways. Furthermore, the formation of GPCRs higher order oligomers provides the structural basis for organizing distinct cell compartments along the plasma membrane where confined increases in second messengers may be perceived and discriminated. Here, we summarize evidence that supports these conjectures, fostering new ideas about the physiological role played by receptor homo- and hetero-oligomerization in cell biology. [ABSTRACT FROM AUTHOR]

(+)-4-Propyl-9-hydroxynaphthoxazine ((+)PHNO) is a high affinity, preferential dopamine D 3 versus D 2 agonist employed in view of its high specificity and excellent signal-to-noise ratio as a radiotracer for positron emission tomography (PET) imaging. Surprisingly, its profile at other classes of monoamine receptor remains undocumented. In addition to hD 3 and hD 2L receptors, (+)PHNO revealed high affinity at hD 4.4 but not hD 1 or hD 5 receptors. It also revealed significant affinity for several other G protein-coupled monoaminergic receptors, in particular h5-HT 1A and h5-HT 7. (+)PHNO behaved as a full agonist at hD 4.4 and h5-HT 1A receptors with potencies comparable to its actions at hD 3 and hD 2L receptors, and with less potency at 5-HT 7 receptors. In binding assays with membranes derived from cells co-expressing hD 3 and hD 2L receptors and labeled with [3H]Nemonapride or [3H]Spiperone, the proportion of high affinity binding sites recognized by (+)PHNO was higher than an equivalent mixture of membranes from cells expressing hD 3 or hD 2L receptors, suggesting that (+)PHNO promotes formation of hD 3 -hD 2L heterodimers. Further, in cells co-expressing hD 3 and hD 2L receptors, (+)PHNO showed higher efficacy for inhibiting forskolin stimulated adenylyl cyclase and inducing adenylyl cyclase super-sensitization than in cells transfected with only hD 2L receptors. In conclusion, (+)PHNO is a potent agonist at hD 4. 4 , h5-HT 1A and h5-HT 7 as well as hD 3 and hD 2L receptors, and it potently activates dopamine hD 3 -hD 2L heterodimers. These interactions should be considered when interpreting PET studies with [11C](+)PHNO and may be relevant to its functional and potential clinical properties in Parkinson's disease and other disorders. [ABSTRACT FROM AUTHOR]