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Post-translational modifications (PTMs) are key events in signal transduction since they affect protein function by regulating their abundance and/or activity. PTMs involve the covalent attachment of functional groups to specific amino acids. Since they tend to be generally reversible, PTMs serve as regulators of signal transduction pathways. G-protein-coupled receptors (GPCRs) are major signaling proteins that undergo multiple types of PTMs. In this Review, we focus on the opioid receptors, members of GPCR family A, and highlight recent advances in the field that have underscored the importance of PTMs in the functional regulation of these receptors. Since opioid receptor activity plays a central role in the development of tolerance and addiction to morphine and other drugs of abuse, understanding the molecular mechanisms regulating receptor activity is of fundamental importance.

Oxytocin (OT) and vasopressin (AVP) are endogenous ligands for OT and AVP receptors in the brain and in the peripheral system. Several studies demonstrate that OT and AVP have opposite roles in modulating stress, anxiety and social behaviours. Interestingly, both peptides and their receptors exhibit high sequence homology which could account for the biased signalling interaction of the peptides with OT and AVP receptors. However, how and under which conditions this crosstalk occurs in vivo remains unclear. In this review we shed light on the complexity of the roles of OT and AVP, by focusing on their signalling and behavioural differences and exploring the crosstalk between the receptor systems. Moreover, we discuss the potential of OT and AVP receptors as therapeutic targets to treat human disorders, such as autism, schizophrenia and drug abuse. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.

Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the ‘opioid epidemic.’ We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling., Lemos Duarte et al. describe a strategy to produce, validate, and utilize highly-specific recombinant antibodies. As a proof of principle, they demonstrate development and validation of a panel of antibodies against proteins relevant to signaling by opiates. Their antibody-based tool can be useful in understanding the spatio-temporal dynamics of opioid signaling.

The enzyme cyclooxygenase 2 (COX-2) is known to be involved in tumorigenesis and metastasis in certain types of cancer. Nevertheless, the prognostic value of COX-2 overexpression and its polymorphisms in patients with non-small cell lung cancer (NSCLC) have yet to be fully elucidated. The aim of the present study was to investigate the association between the three most commonly studied COX-2 gene polymorphisms (−1195 G/A, −765 G/C and 8473 T/C) with COX-2 expression and lung cancer risk in a Brazilian cohort. In the present hospital based, case-control retrospective study, 104 patients with NSCLC and 202 cancer free control subjects were genotyped for −1195 G/A, −765 G/C and 8473 T/C polymorphisms using allelic discrimination with a reverse transcription quantitative polymerase chain reaction method. COX-2 mRNA expression was analyzed in surgically resected tumors from 34 patients with NSCLC. The results revealed that COX-2 expression levels were higher in tumor tissue compared with normal lung tissue. However, this overexpression of COX-2 was not associated with the patient outcome, and furthermore, none of the analyzed polymorphisms were associated with the risk of developing lung cancer, COX-2 overexpression, or the overall survival of the patients with NSCLC. Taken together, the findings described in the present study do not support a major role for COX-2 polymorphisms and COX-2 overexpression in lung carcinogenesis within the Brazilian population.

It is well accepted that treatment of chronic pain with morphine leads to μ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, β-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain.