Abstract
The orphan G protein-coupled receptor (GPCR) GPR3 enhances the processing of Amyloid Precursor Protein (APP) to the neurotoxic beta-amyloid (Aβ) peptide via incompletely understood mechanisms. Through overexpression and shRNA knockdown experiments in HEK293 cells, we show that β-arrestin2 (βarr2), a GPCR-interacting scaffold protein reported to bind γ-secretase, is an essential factor for GPR3-stimulated Aβ production. For a panel of GPR3 receptor mutants, the degree of stimulation of Aβ production correlates with receptor-β-arrestin binding and receptor trafficking to endocytic vesicles. However, GPR3’s recruitment of βarr2 cannot be the sole explanation, because interaction with βarr2 is common to most GPCRs, whereas GPR3 is relatively unique among GPCRs in enhancing Aβ production. In addition to β-arrestin, APP is present in a complex with GPR3 and stimulation of Aβ production by GPR3 mutants correlates with their level of APP binding. Importantly, among a broader selection of GPCRs, only GPR3 and prostaglandin E receptor 2 subtype EP2 (PTGER2; another GPCR that increases Aβ production) interact with APP, and PTGER2 does so in an agonist-stimulated manner. These data indicate that a subset of GPCRs, including GPR3 and PTGER2, can associate with APP when internalized via βarr2, and thereby promote the cleavage of APP to generate Aβ.
Highlights
Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder estimated to affect,5 million people in the United States and approximately 36 million people worldwide, with numbers predicted to grow further as a result of an aging global population [1,2,3]
Knockdown of barr2 lowered basal Ab (0.67+/ 20.04) and suppressed GPR3-induced Ab production (1.07+/ 20.10) (Figure 1D). These data confirm that GPR3 is capable of enhancing Ab production and show that b-arrestin2 is important for this effect in SweAPP-HEK cells
We investigated several mutations in the intracellular domains of GPR3: (i) DRY-AAY, a double point mutant at the base of the third transmembrane domain that impairs the Gs-coupling of many G protein-coupled receptor (GPCR) [31,32,33,34] and reduces GPR3-stimulated cAMP by .85% (Figure S1); (ii) Q302*, a mutant truncated after the seventh transmembrane segment, eliminating the intracellular carboxyterminal tail required for efficient b-arrestin recruitment to most GPCRs [35]; and (iii) S237A, a point mutant in the third intracellular loop which removes a putative G protein-coupled receptor kinases (GRKs) site (Figure 2A)
Summary
Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder estimated to affect ,5 million people in the United States and approximately 36 million people worldwide, with numbers predicted to grow further as a result of an aging global population [1,2,3]. GPR3 exhibits a high constitutive G protein coupling, effects of the receptor on Ab production were independent of Gs and cAMP signaling [14]. It has been shown that b-arrestins coordinate several G protein-independent GPCR signaling cascades [24,25,26]. In these cases, the b-arrestin typically serves as a molecular scaffold, assembling multiple elements of a signaling cascade at activated receptors, thereby regulating the temporal and spatial activity of the pathway. Testing a wider panel of GPCRs, we found all receptors interact with barrestins, but only GPR3 and PTGER2 showed appreciable interaction with APP and stimulated Ab production. We propose that a subset of GPCRs is capable of forming a receptorAPP complex in a barr2-dependent manner to facilitate the generation of Ab
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