Abstract:
The study of arrestins as regulators of seven transmembrane receptor (7TMR)
signaling has revealed multiple levels of complexity, initiating desensitization of G
protein activity and coordination of receptor internalization via clathrin‐coated pits.
Recently, β‐arrestins have also been shown to act as adaptor proteins, mediating G
protein‐independent signaling as well as scaffolding of enzymes that degrade second
messenger molecules. This latter function was demonstrated by β‐arrestins recruiting
PDE4 phosphodiesterase to Gs‐coupled β2‐adrenergic receptors, enhancing metabolism
of the second messenger cAMP. As β‐arrestins universally interact with members of the
7TMR superfamily, we sought to determine if this phenomenon of concerted
desensitization might be applicable to additional receptor subtypes.
We screened for β‐arrestin‐binding proteins among modulators of diacylglycerol
and IP3 (second messengers downstream of Gq‐coupled 7TMRs). We observed β‐
arrestins constitutively interacted with members of the diacylglycerol kinase (DGK)
family, which phosphorylate diacylglycerol to create phosphatidic acid. Furthermore,
examining lipid extracts of 32P labeled cells separated by TLC, we observed that
overexpression of β‐arrestin enhanced phosphatidic acid (PA) production after M1
muscarinic receptor stimulation. Conversely, depletion of β‐arrestins by RNA
interference showed significantly decreased agonist‐stimulated PA accumulation.
Additionally, overexpression of a β‐arrestin2 mutant that binds DGKs but not receptors
served as a dominant negative for agonist‐dependent DGK activity. These results
demonstrate a requirement for β‐arrestins in DGK translocation to the membrane, and
specifically to activated 7TMRs, where concentrations of second messengers are at their
highest.
Phosphatidic acid is an effector for several enzymes, including the
phosphatidylinositol 5‐kinases (PIP5K), which phosphorylate PIP to make PIP2. Thus,
we hypothesized β‐arrestin‐targeted DGKs may regulate PIP5K activity. PIP5K Iα
associated with β‐arrestin2 in an agonist‐dependent manner in HEK293 cells, and a β‐
arrestin2 mutant defective in receptor endocytosis (a PIP2‐dependent function) was
impaired. Furthermore, knockdown of β‐arrestin2 by RNAi significantly decreased the
amount of PIP5K Iα detected in receptor immunoprecipitates. In TLC assays,
overexpressing both β‐arrestin2 and PIP5K Iα enhanced agonist‐stimulated PIP2
labeling, while either protein alone had no effect. These data support the concept of β‐
arrestin binding to 7TMRs and enriching local membrane concentrations of PA, which
then stimulates production of PIP2, promoting receptor internalization.
