G protein receptor kinases (GRKs) and β-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 Ser and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was β-arrestin to a DAMGO-activated MOR. β-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 TREHPSTANT motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and β-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.

No abstract

About ioo years ago, Marchal de Calvi 1 wrote in his first treatise on diabetic gangrene: "Often the opposite leg is affected, gangrene sets in and soon again the patient succumbs to horrible suffering. Having relieved him only of his local affliction (by amputation), I have done nothing but mutilate him." The vast newer literature on lesions of the legs and feet in diabetes testifies to their continued high incidence, seriousness and poor prognosis, but rarely refers to the bilateral lesions. 2 " 5 Yet, the fate of the second leg is intimately related to that of the first leg. Ulcers and gangrene of feet or toes, though precipitated by trauma or other exogenous factors, result from systemic vascular and nervous diseases complicating diabetes. When unilateral lesions call for medical attention, one may well expect involvement of the second leg even though it may appear asymptomatic. The fate of the second leg may reflect the further natural course of the disease; it may be modified by the treatment awarded to the unilateral lesions; and it may deserve serious consideration in deciding on such treatment plan. Evidently there is need for information on this aspect of the complications of •diabetes.As part of a study on peripheral vascular disease in diabetes, we have attempted therefore to assess the integrity or impairment of the second leg in those patients who were admitted to the Jewish Chronic Disease Hospital with unilateral leg lesions or amputations. The study is still in progress, yet a preliminary report on the findings in a first group of admissions during an eighteen-month period appears to be warranted. OBSERVATIONSThere were seventy-one patients. Their age, sex distribution and the time of the development of the first leg lesion in relation to the duration of their diabetes