Cyclic AMP does double duty to support parallel signaling in primary cilium and cytosol

Abstract

The primary cilium maintains all of the necessary machinery to generate and interpret cAMP signals within its tiny volume, leading to the supposition that ciliary cAMP provides unique biological instructions separate from those derived from the rest of the cell body. A new paper by Truong et al. has used optogenetic and chemogenetic tricks to selectively manipulate cAMP signaling within the primary cilium. Their data show that ciliary but not cytosolic message preferentially regulates transcriptional activity via the hedgehog pathway leading to actions on zebrafish development. Computer modeling provides a rational explanation as to how the geometry of this organelle enables it to tune out cAMP signals from the cell body in order to pick up messages generated in the cilium.

One of the enduring mysteries of the primary cilium concerns its propensity to sequester a very specific subset of G-protein-coupled receptors (GPCRs) that are distinct from those found on the rest of the cell body. Vertebrate cilia are small labile structures that sprout from the cell surface when a cell enters quiescence. Their membrane is contiguous with the rest of the plasma membrane, but the proteins that make up the cilium are trapped or trafficked by very specific means. This leads to the assembly of a highly specialized organelle comprised of around 1000 distinct proteins. In contrast, small molecules like second messengers diffuse unhindered between cytoplasm and cilioplasm, reminiscent of the free transfer of small molecules through nuclear pores.

Since the initial descriptions of the ciliary localization of 5-HT₆ and SSTR3 receptors 20+ years ago, more than 30 different ciliary GPCRs have been reported [1,2]. The vast majority of these are coupled to the cAMP signaling pathway via Gαs or Gαi to stimulate or inhibit resident adenylyl cyclases, respectively.

Recent studies using targeted cAMP biosensors have directly demonstrated that 5-HT₆, D1R, FFAR4, and SSTR3 receptors are capable of signaling while in the cilium [3–5]. There is also ample (although somewhat less direct) evidence that the Gαs-coupled orphan receptor GPR161 is operational in the cilium where it impacts another classical ciliary signaling pathway, Hedgehog [6]. Of note, cAMP and PKA have well defined actions on the Gli transcription factors that move between cilium and nucleus to mediate Hedgehog signaling [2]. In a curious twist, our lab recently reported that Hedgehog activation enhanced the ability of ciliary 5-HT₆ and D1R to generate local cAMP signals in primary cilia [3].

A burning question in the field has been whether cAMP generated in the ciliary microdomain provides distinct biological instructions, thereby allowing a single messenger to govern two parallel signaling circuits in cilium and cytosol. While this may be an attractive model, if such a mechanism were to exist how could this concept be reconciled with the fact that small molecules such as calcium and cAMP freely diffuse between the two compartments?

A new proof-of-concept study by Truong et al. used a multi-pronged approach to show that cAMP generated locally within the primary cilium indeed exerts different effects than messenger produced in the cell body [7]. Their first step was to use a cilium-targeted light-activated adenylyl cyclase (cilium-bPAC). When expressed in developing zebrafish embryos, cAMP was generated specifically in the cilium upon exposure to pulsed light, yielding a striking action on Hedgehog-dependent patterning (somite geometry and neural tube formation). This effect, however, was not reproduced by an equivalent amount of cAMP delivered from bPAC localized within the cytoplasm. Experiments in cultured mammalian cells further confirmed that light-stimulated cAMP production via cilium-targeted (but not cytosol-targeted) bPAC could preferentially inhibit Hedgehog signal transduction.

As a complementary strategy to differentially control cAMP generation in the two compartments, the authors used Gαs-coupled DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) targeted to the cilium or plasma membrane. Stimulation of the engineered receptors with the designer drug clozapine-N-oxide caused cAMP to increase in the appropriate location (cilium or cytosol), but again only the ciliary DREADDs were able to efficiently inhibit downstream Hedgehog signaling. The opposite effect was obtained upon stimulation of the native ciliary Gαi-coupled somatostatin receptor, SSTR3.

The preceding suggests that ciliary cAMP has distinct effects on Hh signaling, actions presumably mediated by local PKA (protein kinase A) through regulation of Gli transcription factors in the cilium. To define where PKA was acting in the cell, the authors engineered a dominant negative form of PKA (dnPKA) that they targeted to cilium, cytosol, or the base of the cilium. When expressed in developing zebrafish, the ciliary dnPKA had the most efficient action on Hedgehog-mediated somite patterning compared to the constructs targeted elsewhere in the cell. Furthermore, the presence of cilium-targeted dnPKA was able to specifically negate the effects of cAMP generated with the organelle from photoactivation of ciliary bPAC.

Informative computational models of PKA activation by Truong et al. indicated that the geometry of the cilium, which has a ~13-fold greater surface-to-volume ratio relative to the cell body, could largely explain the preferential activation of PKA in this elongated organelle. It will be interesting in the future to consider recent findings on the biophysical properties of signaling complexes, such as the PKA RIα subunit “droplets” recently described by the group of Jin Zhang [8]. This could further explain the apparent paradox of how the primary cilium maintains a heightened level of signaling efficiency in spite of rapid diffusion of message between compartments. Fig. 1,2.

Fig. 1.

Top panel: 3D volume rendering of live ciliated mIMCD3 cells expressing a cAMP reporter targeted to the primary cilium using the 5-HT₆ serotonin receptor (5-HT₆-EpacH187 [3]; represented in green). Cells have been counterstained with SPYDNA 595 to highlight nuclei (blue), and SiR tubulin to label microtubules (orange; note that SiR tubulin also labels the microtubule core of the cilium, but this is not evident here). The image illustrates the geometry and relatively small size of the cilium and the near perfect localization of the 5-HT₆ receptor to this organelle.

Fig. 2.

Bottom panel: Model of ciliary cAMP circuit. Activated GPCRs couple to cilium-resident Gαs and adenylyl cyclase (e.g. AC3, AC5, or AC6) to generate cAMP locally in the cilium. This engages local PKA, resulting in phosphorylation of Gli3 transcription factor. Gli transits to the cell body where it is converted to a repressor form that inhibits the expression of Hedgehog genes. In order to bypass these native systems, Truong and colleagues used DREADDs in place of ciliary GPCRs and light-activated cyclases (bPAC) in place of ACs to generate cAMP locally, as well as a dominant-negative PKA to selectively block ciliary PKA. Their data indicate that Hedgehog signaling is tuned to preferentially respond to cAMP derived from the ciliary compartment.

Like all breakthrough papers, the elegant work by Truong and colleagues raises more questions than it answers. For example, can GPCRs in the cilium be harnessed to modify Hh-driven cancer progression (e.g. glioblastoma, basal cell carcinoma)? And while hedgehog is classically regulated by ciliary cAMP/PKA, there are likely many other cAMP/PKA targets in the cilium. How might these play into “ciliopathies” like cystic kidney diseases? These findings also highlight our need for a comprehensive catalog of which GPCRs are localized to cilia in particular cell types, and their signaling properties in this environment.

Of interest to the readership of Cell Calcium, the cilium is also a specialized calcium signaling organelle [9]. We are far from fully understanding how calcium ions, with their powerful effects on multiple aspects of cAMP signaling [10], can tune these responses.