GPR88 Reveals a Discrete Function of Primary Cilia as Selective Insulators of GPCR Cross-Talk

Abstract

A number of G protein-coupled receptors (GPCRs) localize to primary cilia but the functional significance of cilia to GPCR signaling remains incompletely understood. We investigated this question by focusing on the D1 dopamine receptor (D1R) and beta-2 adrenergic receptor (B2AR), closely related catecholamine receptors that signal by stimulating production of the diffusible second messenger cyclic AMP (cAMP) but differ in localization relative to cilia. D1Rs robustly concentrate on cilia of IMCD3 cells, as shown previously in other ciliated cell types, but disrupting cilia did not affect D1R surface expression or ability to mediate a concentration-dependent cAMP response. By developing a FRET-based biosensor suitable for resolving intra- from extra- ciliary cAMP changes, we found that the D1R-mediated cAMP response is not restricted to cilia and extends into the extra-ciliary cytoplasm. Conversely the B2AR, which we show here is effectively excluded from cilia, also generated a cAMP response in both ciliary and extra-ciliary compartments. We identified a distinct signaling effect of primary cilia through investigating GPR88, an orphan GPCR that is co-expressed with the D1R in brain, and which we show here is targeted to cilia similarly to the D1R. In ciliated cells, mutational activation of GPR88 strongly reduced the D1R-mediated cAMP response but did not affect the B2AR-mediated response. In marked contrast, in non-ciliated cells, GPR88 was distributed throughout the plasma membrane and inhibited the B2AR response. These results identify a discrete ‘insulating’ function of primary cilia in conferring selectivity on integrated catecholamine signaling through lateral segregation of receptors, and suggest a cellular activity of GPR88 that might underlie its effects on dopamine-dependent behaviors.

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

“…Although a FRET-based cAMP sensor targeted to cilia was previously reported, it was not used to compare cAMP levels between cilia and the whole cell (13). To accurately determine cAMP concentrations, we assayed the response of 5HT6-mCherry-cADDis expressed in MEFs to varying doses of a cell-permeant analog of cAMP, using live-cell imaging, as described previously (21).…”

“…Measuring signaling molecules within cilia has remained challenging due to a paucity of effective tools. A FRET-based cAMP sensor was used to measure the inhibition of D1R signaling by GPR88 in the cilia; however, comparisons between whole-cell and ciliary levels were not made and HH signaling was not assayed (13). A more recent study used a different FRET sensor to study cAMP, but focused mainly on cAMP in flagella (14).…”

An intelligent nano-antenna: Primary cilium harnesses TRP channels to decode polymodal stimuli

“…Dissecting cilium-specific activity from the rest of the cell body requires obtaining adequate ciliary signal-to-background ratio, but this is impeded by the disparate volume ratio between the solitary primary cilium and the cell body. There is hence growing interest to develop tools that can specifically visualize and manipulate activities within this sub-micrometer sized compartment on the cell membrane [7, 60, 61, 73, 74]. …”

Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols

“…The authors proposed an "insulating" function of primary cilia by preventing localization of GPR88 to the plasma membrane, and regulating catecholamine-regulated cellular b2AR signaling (Marley et al, 2013). A better understanding of the interacting partners and posttranslational modifications in GPCRs like phosphorylation, glycosylation, or ubiquitination that affect trafficking, downstream signaling, and sorting is essential.…”

“…In addition, sequestration of certain GPCRs in cilia may also function in reducing localization to the rest of the plasma membrane. In a recent study, GPR88, an orphan ciliary GPCR that is coexpressed in brain neurons with D1R, was examined for modulation of activity of ciliary (D1R) and nonciliary (b2 adrenergic receptor; b2AR) GPCRs (Marley, Choy, & von Zastrow, 2013). In ciliated cells, mutational activation of GPR88 strongly reduced the D1R-mediated cAMP response but did not affect the b2AR-mediated response.…”