Abstract
beta-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 beta-arrestins by using three different methodological
approaches: 1) ability of overexpression of beta-arrestins to restore
the internalization of (125)I-PTH-related protein for the mutant
PTHRs; 2) visualization of PTH-mediated trafficking of beta-arrestin1
and -2 fused to the green fluorescent protein with receptors by confocal
microscopy; 3) quantification of beta-arrestin1-green fluorescent
protein translocation by Western blot. Our data reveal that the receptor'
cytoplasmic tail contains determinants of beta-arrestin interaction
that are distinct from the phosphorylation sites and are sufficient
for transient association of beta-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/beta-arrestin complex toward early endocytic endosomes
during the initial step of endocytosis.
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