Abstract
beta-Arrestins are crucial regulators of G-protein coupled receptor
(GPCR) signaling, desensitization, and internalization. Despite the
long-standing paradigm that agonist-promoted receptor phosphorylation
is required for beta-arrestin2 recruitment, emerging evidence suggests
that phosphorylation-independent mechanisms play a role in beta-arrestin2
recruitment by GPCRs. Several PDZ proteins are known to interact
with GPCRs and serve as cytosolic adaptors to modulate receptor signaling
and trafficking. Na(+)/H(+) exchange regulatory factors (NHERFs)
exert a major role in GPCR signaling. By combining imaging and biochemical
and biophysical methods we investigated the interplay among NHERF1,
beta-arrestin2, and the parathyroid hormone receptor type 1 (PTHR).
We show that NHERF1 and beta-arrestin2 can independently bind to
the PTHR and form a ternary complex in cultured human embryonic kidney
cells and Chinese hamster ovary cells. Although NHERF1 interacts
constitutively with the PTHR, beta-arrestin2 binding is promoted
by receptor activation. NHERF1 interacts directly with beta-arrestin2
without using the PTHR as an interface. Fluorescence resonance energy
transfer studies revealed that the kinetics of PTHR and beta-arrestin2
interactions were modulated by NHERF1. These findings suggest a model
in which NHERF1 may serve as an adaptor, bringing beta-arrestin2
into close proximity to the PTHR, thereby facilitating beta-arrestin2
recruitment after receptor activation.
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