Desensitization Of Receptor Signaling Research Articles

In Vivo Delta Opioid Receptor Internalization Controls Behavioral Effects of Agonists

BackgroundGPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.Methods and FindingsIn this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.ConclusionsTogether our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.

Synucleins Are a Novel Class of Substrates for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (alpha, beta, gamma, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. alpha-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the alpha and beta isoforms, whereas GRK5 prefers alpha-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gbetagamma subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein.

Identification and chromosomal localization of a processed pseudogene of human GRK6

G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G-protein-coupled receptors, resulting in desensitization of receptor signaling. To date, 6 mammalian GRKs have been identified by molecular cloning. Several lines of evidence indicate that a homologue of GRK6, the most recently described GRK, is present in the human genome. Northern analysis identifies two transcripts which hybridize to GRK6, and genomic Southern analysis indicates that GRK6 is localized to chromosome 5, with a second GRK6-like locus on chromosome 13. To identify the GRK6 homologue on chromsome 13, several sets of closely-spaced primers were designed based on the GRK6 cDNA sequence and then used to amplify human genomic DNA by PCR. Two products were identified, the larger of which is a fragment of the GRK6 gene which contains introns, while the smaller fragment is 94% homologous to GRK6 and contains no introns. In order to further characterize this GRK6 homologue, primers from the 5′ and 3′ coding regions of GRK6 were used to amplify a product of 1458 base pairs from human genomic DNA. This 1458 base pair PCR fragment displays 94% homology to GRK6 and contains multiple nucleotide insertions and deletions compared to GRK6, including a C to T mutation at base pair 202 which creates a predicted in-frame stop codon. In an effort to determine whether this gene is transcriptionally active, primers designed to preferentially amplify either GRK6 or the homologue were used in reverse transcription PCR. In contrast to the GRK6-specific primers, primers which selectively amplify the GRK6 homologue fail to produce a PCR product in any RNA tested, indicating that this gene is most likely transcriptionally inactive. PCR amplification of rodent/human hybrid cell lines using these same primers confirms the previously established chromosome 5 localization of GRK6, and localizes this homologue to chromosome 13. Northern analysis indicates that the two GRK6-hybridizing species seen in RNA differ by ∼500 base pairs in the 3′ untranslated region, indicating that both transcripts likely arise from differential processing of a single gene. Taken together, these data indicate that the GRK6-hybridizing species on chromosome 13 is a transcriptionally inactive processed pseudogene of GRK6, while the two GRK6 transcripts differ in the 3′ untranslated region.

Transfected m2 muscarinic acetylcholine receptors couple to G alpha i2 and G alpha i3 in Chinese hamster ovary cells. Activation and desensitization of the phospholipase C signaling pathway.

Muscarinic acetylcholine receptor subtypes (m1-m5) differentially regulate phosphoinositide-specific phospholipase (PLC) through the activation of distinct guanine nucleotide-binding (G) proteins which can be distinguished on the basis of their sensitivity to inhibition by pertussis toxin (PTX). In transfected Chinese hamster ovary cells, the m2 receptor subtype regulates the stimulation of PLC and inhibition of adenylyl cyclase (AC) through PTX-sensitive G proteins. In this study, we utilized the ability of cholera toxin (CTX) to ADP-ribosylate PTX-sensitive alpha subunits as part of the ternary complex formed by heterotrimeric G proteins and agonist-bound receptors to detect and characterize the interactions between transfected m2 receptors and endogenous G proteins in Chinese hamster ovary cells. In membranes derived from cells expressing the m2, but not the m3 receptor, the cholinergic agonist carbachol stimulated CTX modification of a 40-kDa species (G alpha 40). Importantly, similar carbachol dose dependence values and PTX dose sensitivities were observed for m2 receptor-mediated PLC signaling and G alpha 40-CTX modification. High resolution urea-SDS-polyacrylamide gel electrophoresis analysis revealed that G alpha i2 (40 kDa) and G alpha i3 (41 kDa) were components of the G alpha 40 identified by m2 receptor-dependent CTX modification. Furthermore, the sensitivities of G alpha i2 and G alpha i3 to PTX modification were determined to be the same as those for PTX inhibition of G alpha 40 labeling by CTX and m2 receptor-mediated PLC signaling. Similarly, agonist-induced desensitization of m2 receptor-G protein signaling required doses of agonist associated with stimulation of PLC. Desensitization involved receptor sequestration and down-regulation of G alpha i3; however, only the reduction of G alpha i3 required prior activation PLC signaling. Finally, desensitization of m2-G protein coupling could be partially mimicked by treatment with thapsigargin, an inducer of intracellular Ca2+ release, without altering the number of cell surface receptors or G protein levels. These results demonstrate that m2 receptors couple to both G alpha i2 and G alpha i3 in vivo and that this interaction is integral to both positive and negative regulatory pathways leading to activation of PLC and desensitization of receptor signaling.