Internalization Determinants of the Parathyroid Hormone Receptor Differentially Regulate β-Arrestin/Receptor Association

Abstract

␤-Arrestins have been implicated in regulating internalization of the parathyroid hormone receptor (PTHR), but the structural features in the receptor required for this effect are unknown. In the present study performed in HEK-293 cells, we demonstrated that different topological domains of PTHR are implicated in agonist-dependent receptor internalization; truncation of the cytoplasmic tail (PTHR-TR), selective mutations of the cytoplasmic tail to remove the sites of parathyroid hormone (PTH)-stimulated phosphorylation (PTHR-PD), and mutations in the third transmembrane helix (N289A) or in the third cytoplasmic loop (K382A) resulted in a 30 -60% reduction in 125 I-PTH-related protein internalization. To better define the role of these internalization determinants, we have tested the ability of these mutant PTHRs to associate with ␤-arrestins by using three different methodological approaches: 1) ability of overexpression of ␤-arrestins to restore the internalization of 125 I-PTH-related protein for the mutant PTHRs; 2) visualization of PTH-mediated trafficking of ␤-arrestin1 and -2 fused to the green fluorescent protein with receptors by confocal microscopy; 3) quantification of ␤-arrestin1-green fluorescent protein translocation by Western blot. Our data reveal that the receptor' cytoplasmic tail contains determinants of ␤-arrestin interaction that are distinct from the phosphorylation sites and are sufficient for transient association of ␤-arrestin2, but stable association requires receptor phosphorylation. Determinants in the receptor's core (Asn-289 and Lys-382) appear to regulate internalization of the receptor/␤-arrestin complex toward early endocytic endosomes during the initial step of endocytosis.Agonist binding to most G protein-coupled receptors (GPCRs) 1 is quickly followed by the internalization of the agonist-receptor complex into endocytic vesicles. The model developed from studies of the ␤ 2 -adrenergic receptor views internalization as a process facilitated by binding of ␤-arrestin proteins to agonist-activated receptors after phosphorylation of the receptors by G protein-coupled receptor kinases (GRKs) (1, 2). Phosphorylation of GPCRs by GRKs is a prerequisite for the mobilization of cytosolic ␤-arrestins. Binding of ␤-arrestins to GRK-phosphorylated receptors results in the physical uncoupling of receptors from their cognate G proteins and terminates agonist-mediated signaling (3, 4). It was shown recently that ␤-arrestins bind clathrin, a major component of the clathrinbased endocytic machinery, with high affinity and serve as an adaptor that targets activated and phosphorylated receptors to clathrin-coated pits (5-7). In the case of the ␤ 2 -adrenergic receptor, receptor internalization is the consequence of the formation of a complex between ␤-arrestin2, the clathrin adaptor protein AP2, clathrin, and the activated receptor (8). Internalization has at least two outcomes: directing the receptor to a compartment where the phosphates are removed, allowing resensitization, and movement of the r...

Molecular and cellular mechanisms of the anabolic effect of intermittent PTH

“…Indeed, some type of continued PTHR1 signaling in the presence of the hormone seems likely in view of the dramatic differences between the effects of continuous and intermittent PTH elevation on the skeleton, as exemplified by the response of genes like RANKL to the two forms of hormone administration [51]. Recycling of the PTHR1 to the membrane surface following internalization may be one explanation for continued signaling in the presence of the ligand [52].…”

Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism

“…[Aib 1,3 ,M]PTH (1-14) (peptide sequence Aib-V-Aib-E-I-Q-L-M-H-Q-Har-A-K-W-NH 2 ) was kindly provided by T. Gardella (Massachusetts General Hospital and Harvard Medical School, Boston); [Aib 1,3 ,M] refers to the combined modifications of Aib 1,3 , Gln-10, Har 11 , Ala-12, and Trp-14 on human PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), where Aib is the ␣-amino-isobutyric acid and Har is homoarginine (8).…”

“…HEK293 cells were cultivated and transfected as described (11). After selection with 400 g͞ml G418 (Merck), stable cell clones showing membrane expression of the GFP-tagged receptors were selected.…”

“…Extensive studies have demonstrated that [Aib 1,3 , M]PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and other PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) analogs (8,(19)(20)(21) mainly, if not exclusively, bind to the PTHR J-domain in contrast to PTH(1-34), which binds to both receptor's N-and J-domains (22). When [Aib 1,3 , M]PTH (1-14) is bound, it prevents PTH analogs from occupying their binding site at the J-domain of the receptor (8,18).…”

Internalization of REIC/Dkk-3 protein by induced pluripotent stem cell-derived embryoid bodies and extra-embryonic tissues

“…5). It is well known that endocytosis of a ligand-receptor complex occurs within a few minutes (27,28). Internalization of Cy3-REIC was a rapid event, being observed within 5 min after addition of Cy3-REIC (data not shown).…”