RESUMO
G protein receptor kinases (GRKs) and ß-arrestins are key regulators of µ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and ß-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was ß-arrestin to a DAMGO-activated MOR. ß-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370TREHPSTANT379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and ß-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.
Assuntos
Arrestinas/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Regulação da Expressão Gênica , Receptores Opioides mu/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Analgésicos Opioides/farmacologia , Arrestinas/química , Quinase 2 de Receptor Acoplado a Proteína G/química , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação , Fosforilação/efeitos dos fármacos , Receptores Opioides mu/agonistas , Homologia de Sequência , Serina/genética , Serina/metabolismo , Transdução de Sinais , Treonina/genética , Treonina/metabolismoRESUMO
Male gender predisposes to severe sepsis and septic shock. This effect has been ascribed to higher levels of testosterone. The ESPNIC ARDS database was searched, to determine if there was evidence of a similar male preponderance in severe sepsis in prepubertal patients in spite of low levels of male sex hormones at this age. A total of 72 patients beyond neonatal age up to 8 years of age with sepsis were identified. The male/female (M/F) ratio was 1.7 (1.0;2.7) and differed significantly from non-septic ARDS patients in this age group [n = 209; M/F = 1.0 (0.8;1.3)]. The highest M/F-ratio was observed in the first year of life. The gender-ratio was the same as reported in adult patients with sepsis. In infants between 1 month and 12 months of age, the ratio was 2.8 (1.2;6.1) (Chi2= 5.6; P< 0.01), in children from 1 year to 8 years of age it was 1.2 (0.7;2.2) (n.s.). In a subgroup of patients with severe sepsis or septic shock, caused by other bacteria than Neisseria meningitidis, the M/F-ratio was 2.1 (1.2;3.6) (Chi2= 4.9; P<0.05), while in patients with meningococcal sepsis (n=20) the M/F-ratio was 1.0 (0.4;2.3). In prepubertal ARDS patients with sepsis an increased frequency of male patients is found, comparable to adults. No male preponderance exists in patients with ARDS due to meningococcal septic shock. Since levels of testosterone and other sex hormones are extremely low at this age, we conclude that factors others than testosterone are involved in the male preponderance in severe sepsis.
