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Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

OBJECTIVE: To examine differences in morbidity and mortality due to non-insulin dependent diabetes in African Caribbeans and Europeans. DESIGN: Cohort study of patients with non-insulin dependent diabetes drawn from diabetes clinics in London. Baseline investigations were performed in 1975-7; follow up continued until 1995. PATIENTS: 150 Europeans and 77 African Caribbeans with non-insulin dependent diabetes. MAIN OUTCOME MEASURES: All cause and cardiovascular mortality; prevalence of microvascular and macrovascular complications. RESULTS: Duration of diabetes was shorter in African Caribbeans, particularly women. African Caribbeans were more likely than the Europeans to have been given a diagnosis after the onset of symptoms and less likely to be taking insulin. Mean cholesterol concentration was lower in African Caribbeans, but blood pressure and body mass index were not different in the two ethnic groups. Prevalence of microvascular and macrovascular complications was insignificantly lower in African Caribbens than in Europeans. 59 Europeans and 16 African Caribbeans had died by the end of follow up. The risk ratio for all cause mortality was 0.41 (95% confidence interval 0.23 to 0.73) (P = 0.002) for African Caribbeans v Europeans. This was attenuated to 0.59 (0.32 to 1.10) (P = 0.1) after adjustment for sex, smoking, proteinuria, and body mass index. Further adjustment for systolic blood pressure, cholesterol concentration, age, duration of diabetes, and treatment made little difference to the risk ratio. Unadjusted risk ratio for cardiovascular and ischaemic heart disease were 0.33 (0.15 to 0.70) (P = 0.004) and 0.37 (0.16 to 0.85) (P = 0.02) respectively. CONCLUSIONS: African Caribbeans with non-insulin dependent diabetes maintain a low risk of heart disease. Management priorities for diabetes developed in one ethnic group may not necessarily be applicable to other groups.

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.