Different mechanisms of homologous and heterologous μ-opioid receptor phosphorylation

The efficiency of μ-opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific μ-opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist-induced phosphorylation of μ-opioid receptors occurs at a conserved 10 residue sequence ³⁷⁰TREHPSTANT³⁷⁹ in the receptor''s carboxyl-terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high-efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist-independent heterologous μ-opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite-specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high-efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of G_q-coupled receptors is mediated by PKCα. Rapid μ-opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ-opioid receptor phosphorylation. However, it remains to be seen to what extent different μ-opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Maturational gonadotropin from the African catfish, Clarias gariepinus: purification, characterization, localization, and biological activity.

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.     

A recalcitrant, erythematous, desquamating disorder associated with toxin-producing staphylococci in patients with AIDS.

Although staphylococcal infections are common in patients with AIDS, staphylococcal toxin-related disorders have rarely been described. Five cases of a staphylococcal toxin-associated syndrome characterized by prolonged erythema, extensive cutaneous desquamation, hypotension, tachycardia, and multiple organ involvement are described in patients with AIDS. These illnesses were recurrent and recalcitrant with a mean duration of 50 days. Toxic shock syndrome toxin-1-producing staphylococci were isolated from three and staphylococcal enterotoxins B and A from one patient each. Sources of organisms were blood, one patient, and soft tissues and nasal accessory sinuses, two patients each. Three of the five patients died of renal failure and central nervous system abnormalities. One survivor required intubation for respiratory failure. All individuals manifested a marked diminution of CD4+ cells. Other laboratory abnormalities included azotemia and prolongation of partial thromboplastin time. Oliguria occurred in three patients. Thus, this recalcitrant erythematous desquamative disorder appears to be a variant of staphylococcal toxic shock syndrome in certain subsets of immunocompromised individuals.     

Regulation and cellular localization of the membrane-bound thyrotropin-releasing hormone-degrading enzyme in primary cultures of neuronal, glial and adenohypophyseal cells

Using monolayer cultures from murine brain and reaggregate cell cultures of rat anterior pituitary we observed that TRH (pyroGlu-His-Pro-NH2) added to the culture medium was not taken up by these cells but hydrolyzed at the pyroGlu-His bond by an enzyme obviously located at the cell surface. This enzyme exhibited a high degree of substrate specificity and other characteristics of the membrane-bound TRH-degrading enzyme. Relatively high enzymatic activity was associated with cultured neuronal cells from embryonic rat brain while glial cells were almost devoid of this peptidase activity. Rather low, but significant activity was found on anterior pituitary cell aggregates. In agreement with previous in vivo studies we observed that the TRH-degrading ectoenzyme on adenohypophyseal cells was regulated by estradiol and stringently controlled by T3, but that the activity of the brain enzyme was not. When pituitary cells were separated according to their size and density and established in reaggregate cell culture, a close correlation was always observed between enzyme activity and the distribution of lactotrophic cells regardless of the animal models (eu- and hypothyroid adult male rats) used and the cell fractionation techniques (velocity sedimentation and sequential velocity/buoyant density sedimentation) employed. Such a close correlation was not observed with other cell types, such as the somatotrophic cells, the folliculo-stellate cells, the ACTH-producing AtT20 pituitary cells, or thyrotrophic cells. In conclusion, the high degree of substrate specificity, the tissue-specific regulation, and the very heterogeneous distribution of the TRH-degrading ectoenzyme on brain and pituitary cells strongly support the hypothesis that this enzyme serves very specialized functions in the transmission of TRH signals at specific target sites.     

Possibilities and results of "immunotherapy" in children with acute leucosis (author's transl)

In the introduction we are giving an overview about the possibilities of treatment of acute leucemias in childhood with an adjuvant immunotherapeutic component. Our own ten-years-experiences from 25 a. 1.1. children with BCG-application are useful for testing this kind of adjuvant therapy under clinical conditions in future, too.     

Course of pulmonary hemodynamics in intermittent ventilation

Patients with chronic respiratory failure often show an elevated pulmonary vascular resistance. We examined in a prospective study with the follow-up of patients with intermittent non-invasive ventilation (nISB) and pulmonary hypertension using dobutamine stress echocardiography. The data show that ISB lowers the pressure in all patients, more in patients with restrictive diseases, due to thoracic deformities, than in patients with COPD.     

Ventilatory failure in COPD: follow-up under intermittent positive pressure ventilation (IPPV)

Effectiveness of IPPV in COPD is controversial. We examine the course of 29 patients with longterm IPPV (19 male, 10 female, age 60.3 years, BMI 23.7, FEV1 mean 22% predicted, PaCO2 mean 67 mmHg). PaCO2 is significantly by IPPV reduced. Patients are followed for 2 to 48 months (mean 16.5). Five stop IPPV (1 bilateral lung transplantation, 1 lung volume reduction surgery, 3 non-compliance), 8 die of respiratory failure, 5 of non-respiratory causes (non-RI) (3 tumor, 2 cardiovascular), and 10 survive (SV, 20.5 months). No difference in survival is observed between non-RI and SV. Weight increase by +5% is seen in SV more frequently, lung function is worse in RI, especially in weaning pts., hospital days are less frequent in SV and non-RI. Probability of survival is 70% at 1 year, 57% at 2 years, and 23% at 3 years, and is seriously influenced by non-RI. Our results are influenced by the high number of non-RI, the quitting of IPPV, and the primary inclusion of tumour pts. Still a high number of deaths by RI leads us to the conclusion that IPPV may be helpful for palliation, bridging before surgery and in subgroups who still have to be defined.