Interrogating cyclic AMP signaling using optical approaches

Abstract

Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.

Keywords: Epac; FRET; Fluorescent proteins; PKA; cAMP signaling microdomains.

Figure 1. Evolution of cAMP sensors

Cartoon describes the genealogy of cAMP sensors originating from PKA, Epac, CNGCs and MlotiK1.

Figure 2. Measurement of cAMP using a biosensor targeted to the primary cilium in NIH-3T3 cells

This example illustrates some of the difficulties in measuring cAMP using organelle-targeted probes. The reporter, ICUE3-SSTR3 (a kind gift of Mark von Zastrow [71]) is based on a fusion between the ciliary GPCR, somatostatin receptor 3 (SSTR3) and ICUE3, an improved cAMP sensor engineered by Jin Zhang’s lab [44]. The fluorescence appears to be quite concentrated within the cilium (arrow), but in this particular case it is actually only twice as great as the non-targeted probe in the plasma membrane. By the criteria of Geneva and colleagues [72], the probe, in spite of appearances, is likely no more concentrated in the cilium than in the plasma membrane (see text for details). Ratiometric FRET measurements show that both ciliary and extra-ciliary regions respond to the membrane permeable cAMP analog, 8-Br-cAMP-AM, with a very small additional increase following addition of the PDE inhibitor IBMX and AC activator forskolin. FRET measurements were performed as described previously [76](Scale bar: 10 μm).