β-Arrestin Mediates Desensitization and Internalization but Does Not Affect Dephosphorylation of the Thyrotropin-releasing Hormone Receptor

Abstract

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to ␤-arrestin after agonist exposure. To define the importance of receptor phosphorylation and ␤-arrestin binding in desensitization, and to determine whether ␤-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking ␤-arrestin-1 and/or ␤-arrestin-2. Apparent affinity for [ 3 H]MeTRH was increased 8-fold in cells expressing ␤-arrestins, including a ␤-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of ␤-arrestins, but receptors remained primarily on the plasma membrane without ␤-arrestin. ␤-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous ␤-arrestins reduced TRH-stimulated inositol phosphate production by 48% (␤-arrestin-1), 71% (␤-arrestin-2), and 84% (␤-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by ␤-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without ␤-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking G␣ q and G␣ 11 or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that ␤-arrestin binding is critical for desensitization and internalization.The type 1 thyrotropin-releasing hormone (TRH) 2 receptor is a seven-transmembrane helix protein that regulates thyroid stimulating hormone and prolactin release from the anterior pituitary. Upon exposure to agonist, the TRH receptor stimulates phospholipase C␤ through coupling to the GTP-binding proteins G␣ q or G␣ 11 , leading to the formation of inositol 1,4,5-trisphosphate (IP 3 ) and the subsequent release of Ca 2ϩ from the endoplasmic reticulum. This signaling pathway ultimately leads to the release of thyroid stimulating hormone and is essential for proper thyroid function and targeted deletion of the type 1 TRH receptor results in hypothyroidism (1).When activated, most G protein-coupled receptors (GPCRs) undergo phosphorylation, which is followed by receptor interaction with ␤-arrestins and desensitization and endocytosis (2). Phosphorylation is carried out by second messenger-activated kinases, such as protein kinase C, or by G protein-coupled receptor kinases (GRKs). Less is known about dephosphorylation, but it is believed to be a crucial step in the resensit...

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

“…When GRK2 was overexpressed, TRH-dependent phosphorylation was increased (Fig. 6A), confirming previous evidence that TRH receptor phosphorylation is primarily because of GRKs (14,17,20). Multiple lines of evidence show that the TRH-dependent phosphorylation observed with the coexpression of D71A and 4Ala-TRH receptors is not because of second messenger-activated kinases.…”

G‐protein‐coupled receptor dephosphorylation at the cell surface

“…Apparently it does; a recent study (Gardiner et al, 2001) shows that the agonist-phosphorylated G s -coupled D 1 dopamine receptor undergoes the same rate of dephosphorylation whether or not it is able to undergo internalization. Furthermore, a very recent study shows that the agonist-activated G q -coupled thyrotropin-releasing hormone receptor is rapidly dephosphorylated at the cell membrane in mouse embryo fibroblast cells from arrestin-knockout animals where these receptors are unable to undergo significant internalization (Jones & Hinkle, 2005).How to reconcile these recent findings with the earlier results (Yu et al, 1993;Pippig et al, 1995;Krueger et al, 1997)? Iyer et al (2005 suggest that the interpretation of earlier studies on the b 2 -adrenoceptor is made difficult because the 32 P labelling technique reflects a combination of PKA and GRK phosphorylation of the receptor.…”

“…Apparently it does; a recent study (Gardiner et al, 2001) shows that the agonist-phosphorylated G s -coupled D 1 dopamine receptor undergoes the same rate of dephosphorylation whether or not it is able to undergo internalization. Furthermore, a very recent study shows that the agonist-activated G q -coupled thyrotropin-releasing hormone receptor is rapidly dephosphorylated at the cell membrane in mouse embryo fibroblast cells from arrestin-knockout animals where these receptors are unable to undergo significant internalization (Jones & Hinkle, 2005).…”

Lysophospholipids of Different Classes Mobilize Neutrophil Secretory Vesicles and Induce Redundant Signaling through G2A

“…Such observations are typical of GPCRs. However, unlike many studies of GPCR desensitization (45)(46)(47), G2A desensitization was not accompanied by obvious loss of the receptor from the cell surface; rather, desensitization to lyso-PL stimulation was temporally associated with visible G2A patching (Fig. 8), perhaps the result of progressive receptor self-association (Fig.…”

“…8; lyso-PC stimulation shown). Although receptor internalization has been reported as accompanying desensitization of many GPCRs following stimulation (45)(46)(47), the maintenance of elevated surface G2A through the period of functional desensitization suggested that G2A desensitization in neutrophils is more clearly associated with patching of the receptor than to its internalization. As such, we reasoned that any stimulus mobilizing secretory vesicles and patching G2A would lead to a nonresponsive state to subsequent lyso-PL stimulation.…”