Role of Arrestins in Endocytosis and Signaling of α2-Adrenergic Receptor Subtypes

Abstract

We investigated the role of arrestins in the trafficking of human ␣ 2 -adrenergic receptors (␣ 2 -ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. ␣ 2 -ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with ␣ 2c > ␣ 2b > ␣ 2a . However, upon coexpression of arrestin-2 (␤-arrestin-1) or arrestin-3 (␤-arrestin-2), internalization of the ␣ 2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the ␣ 2c AR was selectively promoted by coexpression of arrestin-3, while ␣ 2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any ␣ 2 -AR subtype, either in the presence or absence of arrestins. Internalization of the ␣ 2b and ␣ 2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, ␣ 2 -AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/ p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and ␤ 2 -adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the ␣ 2b , ␣ 2c , and to a lesser extent ␣ 2a ARs, is both arrestin-and dynamin-dependent. However, endocytosis does not appear to be required for ␣ 2 -adrenergic, epidermal growth factor, lysophosphatidic acid, or ␤ 2 -adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.

Trafficking of the HIV Coreceptor CXCR4

“…In contrast, CXCR4 internalization was efficient in the absence of GRK2 and arrestin overexpression in both HEK-293 and COS-1 cells. COS cells contain lower levels of endogenous GRKs and arrestins, and internalization of both ␣-and ␤-adrenergic receptors in COS cells is inefficient in the absence of overexpressed arrestins (47,50,51,68). However, SDF-1␣-mediated internalization of CXCR4 in COS cells was equivalent to that observed in HEK-293 cells, although PMA-mediated internalization was less efficient.…”

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

“…Prolonged incubation of the cells with UK14304 did not influence ␣ 2 -AR expression or [ 125 I]VIP binding at the cell surface. Incubation of cells with the ␣ 2 -AR agonist (18 h) also did not alter the ability of the ␣ 2 -AR to mediate inhibition of VIP-induced increases in cAMP, 5 reflecting the limited ability of this ␣ 2 -AR subtype to undergo desensitization and down-regulation (36,37).…”

New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades

“…Endocytosis has also been shown to be important for ERK activation by the 5HT 1A receptor (Della Rocca et al, 1999b), the m1 muscarinic receptor (Vogler et al, 1999), and the d, k and m opioid receptors (Ignatova et al, 1999;Whistler and von Zastrow, 1999). On the other hand, inhibiting clathrin-mediated internalization has been shown not to a ect ERK activation mediated by the a 2A,B , and C adrenergic receptors (DeGra et al, 1999;Schramm and Limbird, 1999), the k opioid receptor (Li et al, 1999), the m3 muscarinic receptor (Budd et al, 1999) and others (Blaukat et al, 1999;Roche et al, 1999;Yang et al, 1999). The fact that the same receptor expressed in di erent cellular contexts can mediate ERK activation that is alternatively endocytosis dependent or independent argue for the existence of heterogeneity in signaling mechanisms.…”