Stefan Schulz

The Conserved Bardet-Biedl Syndrome Proteins Assemble a Coat that Traffics Membrane Proteins to Cilia

The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII, and clathrin coats. Here, we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. The BBSome is the major effector of the Arf-like GTPase Arl6/BBS3, and the BBSome and GTP-bound Arl6 colocalize at ciliary punctae in an interdependent manner. Strikingly, Arl6(GTP)-mediated recruitment of the BBSome to synthetic liposomes produces distinct patches of polymerized coat apposed onto the lipid bilayer. Finally, the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome in order to mediate targeting of membrane proteins to cilia. Thus, we propose that trafficking of BBSome cargoes to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals by the BBSome.

Regulation of μ-opioid receptors: Desensitization, phosphorylation, internalization, and tolerance

Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance.

Homo- and Heterodimerization of Somatostatin Receptor Subtypes INACTIVATION OF sst3 RECEPTOR FUNCTION BY HETERODIMERIZATION WITH sst2A

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst2A and sst3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst2A and sst3. The sst2A-sst3 heterodimer exhibited high affinity binding to somatostatin-14 and the sst2-selective ligand L-779,976 but not to the sst3-selective ligand L-796,778. Like the sst2A homodimer, the sst2A-sst3 heterodimer stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst2-selective ligand L-779,976. However, unlike the sst3 homodimer, the sst2A-sst3 heterodimer did not promote GTPγS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst3-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst2A-sst3 heterodimer desensitized at a slower rate than the sst2A and sst3 homodimers. Both sst2A and sst3 homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst2A-sst3 heterodimer separated at the plasma membrane, and only sst2A but not sst3 underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst2A and sst3results in a new receptor with a pharmacological and functional profile resembling that of the sst2A receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst3 receptor function by heterodimerization with sst2A or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst3-specific binding and signaling in mammalian tissues.

Effect of the A118G polymorphism on binding affinity, potency and agonist‐mediated endocytosis, desensitization, and resensitization of the human mu‐opioid receptor

The most prevalent single‐nucleotide polymorphism (SNP) A118G in the human µ‐opioid receptor gene predicts an amino acid change from an asparagine residue to an aspartatic residue in amino acid position 40. This N40D mutation, which has been implicated in the development of opioid addiction, was previously reported to result in an increased β‐endorphin binding affinity and a decreased potency of morphine‐6‐glucuronide. Therefore, in the present study we have investigated whether this mutation might affect the binding affinity, potency, and/or the agonist‐induced desensitization, internalization and resensitization of the human µ‐opioid receptor stably expressed in human embryonic kidney 293 cells. With the exception of a reduced expression level of N40D compared to human µ‐opioid receptor (hMOR) in HEK293 cells, our analyses revealed no marked functional differences between N40D and wild‐type receptor. Morphine, morphine‐6‐glucuronide and β‐endorphin revealed similar binding affinities and potencies for both receptors. Both the N40D‐variant receptor and hMOR exhibited robust receptor internalization in the presence of the opioid peptide [d‐Ala2,N‐MePhe4,Glyol5]enkephalin (DAMGO) and β‐endorphin but not in response to morphine or morphine‐6‐glucuronide. After prolonged treatment with morphine, morphine‐6‐glucuronide or β‐endorphin both receptors showed similiar desensitization time courses. In addition, the receptor resensitization rates were nearly identical for both receptor types.

Carboxyl-terminal Splicing of the Rat μ Opioid Receptor Modulates Agonist-mediated Internalization and Receptor Resensitization

The rat μ opioid receptor is alternatively spliced into two isoforms (MOR1 and MOR1B) which differ in length and amino acid composition at the carboxyl terminus. When stably expressed in HEK 293 cells, both splice variants bind the μ receptor agonist [d-Ala2,N-Me-Phe4,-Gly-ol5]enkephalin (DAMGO) with similar affinity and exhibit functional coupling to adenylyl cyclase with similar efficiency. However, the shorter isoform, MOR1B, desensitized at a slower rate during prolonged DAMGO exposure (4 h) but resensitized at a faster rate than MOR1 during agonist withdrawal (20 min). Immunocytochemical analysis revealed that DAMGO-induced internalization of MOR1B proceeded much faster than that of MOR1 followed by rapid recycling of the receptor to the cell surface. In addition, the greater resistance of MOR1B to homologous desensitization compared with MOR1 as well as MOR1B resensitization was abolished when receptor reactivation/recycling was blocked with monensin, an inhibitor of endosomal acidification. It is concluded that the sequence at the cytoplasmic tail of MOR1B facilitates clathrin-coated vesicle-mediated endocytosis which, in turn, promotes accelerated receptor reactivation. Taken together, our findings suggest that carboxyl-terminal splicing of the rat μ opioid receptor modulates agonist-induced internalization and receptor resensitization.

Cxcr7 Controls Neuronal Migration by Regulating Chemokine Responsiveness

The chemokine Cxcl12 binds Cxcr4 and Cxcr7 receptors to control cell migration in multiple biological contexts, including brain development, leukocyte trafficking, and tumorigenesis. Both receptors are expressed in the CNS, but how they cooperate during migration has not been elucidated. Here, we used the migration of cortical interneurons as a model to study this process. We found that Cxcr4 and Cxcr7 are coexpressed in migrating interneurons, and that Cxcr7 is essential for chemokine signaling. Intriguingly, this process does not exclusively involve Cxcr7, but most critically the modulation of Cxcr4 function. Thus, Cxcr7 is necessary to regulate Cxcr4 protein levels, thereby adapting chemokine responsiveness in migrating cells. This demonstrates that a chemokine receptor modulates the function of another chemokine receptor by controlling the amount of protein that is made available for signaling at the cell surface.

Identification of somatostatin receptor subtypes 1, 2A, 3, and 5 in neuroendocrine tumours with subtype specific antibodies

Background and aims: Recently, novel somatostatin receptor (sstr) subtype specific ligand analogues have been developed for medical treatment of neuroendocrine tumours expressing different sstrs (sstr1–5). At present, individual expression patterns of sstr subtypes are based on methods such as in situ hybridisation and polymerase chain reaction at the transcriptional level. Therefore, we generated subtype specific antibodies against sstr1, 2A, 3, and 5 and analysed their presence, cellular localisation, distribution, and expression pattern in 33 gastrinomas, 36 insulinomas, and 35 tumours associated with a carcinoid syndrome by immunohistochemistry at the translational level. Methods: Western blotting experiments were performed in the normal human pancreas used as a reference organ and in tumour tissues; at the cellular level, sstrs were localised by immunohistochemistry in tissue paraffin sections. Results: In western blot analyses, the antibodies identified the respective receptors in their correct molecular range in extracts of the pancreas and neuroendocrine tumours. Using immunohistochemistry and immunofluorescence, the antibodies specifically detected the receptors in islet cells of the normal pancreas. Immunohistochemistry in the tumours revealed that all investigated sstr subtypes were highly expressed in the different tumour types. The frequency and expression pattern of the individual sstr subtypes varied considerably not only between the different tumour types but also in each patient. Conclusions: We conclude that immunohistochemistry with subtype specific antibodies can be used in clinical routine work to analyse sstr expression patterns for each patient before treatment and to facilitate well directed individual medical therapy by administering subtype specific somatostatin analogues.

Morphine induces terminal μ-opioid receptor desensitization by sustained phosphorylation of serine-375

Morphine is a poor inducer of μ‐opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D‐Ala2‐MePhe4‐Gly‐ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxy‐terminal residue 375 (Ser375). Ser375 phosphorylation was sufficient and required for morphine‐induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO‐desensitized receptors were rapidly dephosphorylated. In contrast, morphine‐desensitized receptors remained at the plasma membrane in a Ser375‐phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser375.

Heterodimerization of Substance P and μ-Opioid Receptors Regulates Receptor Trafficking and Resensitization

The μ-opioid receptor (MOR1) and the substance P receptor (NK1) coexist and functionally interact in nociceptive brain regions; however, a molecular basis for this interaction has not been established. Using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that MOR1 and NK1 can form heterodimers in HEK 293 cells coexpressing the two receptors. Although NK1-MOR1 heterodimerization did not substantially change the ligand binding and signaling properties of these receptors, it dramatically altered their internalization and resensitization profile. Exposure of the NK1-MOR1 heterodimer to the MOR1-selective ligand [d-Ala2,Me-Phe4,Gly5-ol]enkephalin (DAMGO) promoted cross-phosphorylation and cointernalization of the NK1 receptor. Conversely, exposure of the NK1-MOR1 heterodimer to the NK1-selective ligand substance P (SP) promoted cross-phosphorylation and cointernalization of the MOR1 receptor. In cells expressing MOR1 alone, β-arrestin directs the receptors to clathrin-coated pits, but does not internalize with the receptor. In cells expressing NK1 alone, β-arrestin internalizes with the receptor into endosomes. Interestingly, in cells coexpressing MOR1 and NK1 both DAMGO and SP induced the recruitment of β-arrestin to the plasma membrane and cointernalization of NK1-MOR1 heterodimers with β-arrestin into the same endosomal compartment. Consequently, resensitization of MOR1-dependent receptor functions was severely delayed in coexpressing cells as compared with cells expressing MOR1 alone. Together, our findings indicate that MOR1 by virtue of its physical interaction with NK1 is sequestered via an endocytotic pathway with delayed recycling and resensitization kinetics.

Nociceptin/orphanin FQ and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions.

ANTISERA were generated against nociceptin/orphanin FQ, the putative ligand of the opioid receptor-like ORL1 receptor. Dot blot analysis showed that the antibodies selectively detect nociceptin but not dynorphin or other opioid peptides. Immunofluorescent staining of tissue sections revealed dense plexus of nociceptin-immunoreactive nerve fibres and terminals within the spinal cord dorsal horn, sensory trigeminal complex, raphe nuclei, locus coeruleus, periaqueductal grey, amygdala, habenula, hypothalamic region and septal area in mice and rats. When adjacent sections were stained either with the nociceptin antibody or the pan-opioid 3-E7 mouse monoclonal antibody, an overlapping distribution was observed in many nociceptive centres including the superficial dorsal horn, sensory trigeminal complex and periaqueductal grey. However, confocal microscopic examination of dual-labelled spinal cord and brain stem sections showed no instances of co-localization of nociceptin and opioid peptides in these regions. Intra- cerebroventricular administration of nociceptin has been shown to induce hyperalgesia. Thus, the present results suggest that nociceptin and opioids are released from different terminals thereby modulating pain signals in opposite ways.