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We describe methods based on live cell fluorescent microscopy and mass spectrometry to characterize the mechanism of endosomal cAMP production and its regulation using the parathyroid hormone (PTH) type 1 receptor as a prime example. These methods permit to measure rapid changes of cAMP levels in response to PTH, kinetics of endosomal ligand–receptor interaction, pH changes associated with receptor trafficking, and to identify the endosomal receptor interactome.
GPCR constitutes the largest family of cell surface transmembrane receptors that transmit extracellular signals into cells. They are involved in multiple aspects of human health and diseases and are prime pharmacological targets for drug discoveries. Many GPCRs transduce their signals by stimulating the production of cAMP, which is needed to regulate major physiological functions. Once believed to be exclusively generated at the plasma membrane (Pierce, Premont, & Lefkowitz, 2002), cAMP can also be produced from endosomes after receptor internalization (Vilardaga, Jean-Alphonse, & Gardella, 2014). The mechanism and biological relevance of this new mode of cAMP signaling become to be understood for some receptors such as the parathyroid hormone (PTH) receptor (PTHR) and the vasopressin V2 receptor (Cheloha, Gellman, Vilardaga, & Gardella, 2015; Feinstein et al., 2013; Gardella & Vilardaga, 2015). The regulation of these altered modes of cAMP signaling (plasma vs endosomal membranes) has been, at least in part, recently uncover for the PTHR (Figure 1). In brief, ligand–receptor (L-R) signaling complexes localized at the plasma membrane induce transient cAMP responses that are mainly terminated by the action of cAMP-specific phosphodiesterases (PDE), whereas prolonged cAMP responses are derived from complexes associated within endosomes. Unexpectedly, the internalized L-R signaling complexes contain β-arrestin1 or β-arrestin2, which promotes, rather than terminates, cAMP signaling by activating ERK1/2, leading to the inhibition of PDE4 activity and sustained cAMP signaling (Ferrandon et al., 2009). Termination of endosomal signaling is initiated by a negative feedback loop where PTH-mediated PKA activation leads to v-ATPase phosphorylation and subsequent endosomal acidification, resulting in the disassembly of signaling L-R–arrestin complexes and assembly of inactive receptor–retromer complexes (Gidon et al., 2014), which sort the receptor to retrograde trafficking domains (Feinstein et al., 2013). This chapter describes several methods that permit to investigate intracellular endosomes-associated GPCR signaling.
The principle of FRET and its use as a tool to study kinetics along the individual biochemical events of the GPCR-signaling cascade in live cells has been previously reviewed (Vilardaga et al., 2009). Here we used FRET to study receptor–ligand interactions or changes in second messenger (cAMP) production in live cells. We describe methods to record FRET in live cells using either wide-field, total internal reflection fluorescence (TIRF) or confocal fluorescence microscopes.
The FRET ratio for single cell experiments is calculated according to Eqn (1)
where FYFP (ex436/em535) and FCFP (ex436/em480) represent the emission intensities of YFP (recorded at 535 nm) and CFP (recorded at 480 nm), respectively, upon excitation at 436 nm; a and b represent correction factors for the bleed-through of CFP into the 535 nm channel (a = 0.35) and the cross talk due to the direct YFP excitation by light at 436 nm (b = 0.06). FYFP (ex500/em535) represents the emission intensity of YFP (recorded at 535 nm) upon direct excitation at 500 nm and was recorded at the beginning of each experiment. Note that the bleed-through of YFP into the 480 nm channel is negligible in our recording system. For each measurement, changes in fluorescence emissions due to photobleaching are subtracted. To ensure that CFP- and YFP-labeled molecule expressions are similar in examined cells; experiments are performed in cells displaying comparable fluorescence levels. FRET data are normalized for different expression levels of CFP and YFP molecules according to Eqn (2),
Generation of cAMP in cells stably expressing PTHR is analyzed in real time using the FRET-based sensor EPAC-CFP/YFP (10) both in wide-field and in TIRF (Figure 2(A) and (B)).
FRAP experiments to determine the stability of receptor–arrestin complexes localized on endosomes have been previously described (Vilardaga, Romero, Feinstein, & Wehbi, 2013). Here we described a method to record interactions of PTHR with either β-arrestin1 or a retromer complex subunit using by real-time wide-field and confocal FRET.
Localization of PTHR and PTH monitored by real-time confocal imaging (Figure 3(A) and (B)).
pH measurement along the endocytic pathway is based on FITC sensitivity to protonation (Lanz, Gregor, Slavík, & Kotyk, 1997).
Data obtained after spectral deconvolution are analyzed as follows: (1) a region of interest is drawn around each single cell; (2) respective FITC and CFP fluorescence levels are recorded; (3) the ratio FFITC/FCFP is plotted over time; (4) the ratio FFITC/FCFP is normalized to the values for t = 1; (5) pH is estimated by comparison with the linear relationship between FITC fluorescence and pH obtained in Section 2.4.1.
Note 1: Both the pulse with PTH(1–34) and the chase with FRET buffer are done while the acquisition is running. These two steps need to be executed carefully in order to keep the focus plane.
Note 2: CFP emission allows to normalize the variation of FITC due to both variation in total amount of available fluorophores and variation due to focus changes induce by endosomes movements.
Measuring the internalization of PTHR and transfer from endosomal domains labeled with β-arrestin1 to domains labeled with Vps35 retromer subunit. These experiments are performed in live cells to avoid the loss of endosomal domain structure that normally happens upon fixation.
Endosomal signaling is a new concept in GPCR biology whereby internalized receptors continue to stimulate the production of cAMP via Gs. Methods discussed in this chapter provide direct access to spatiotemporal analyses of GPCR signaling at the single cells level and permit to investigate molecular and cellular mechanisms that govern GPCR signaling when they redistribute in endosomes. In the case of the PTHR, we previously demonstrated that this receptor (R) adopts at least two distinct signaling conformations, R0 and RG (Vilardaga et al., 2014). R0-selective ligands (such as PTH) prolong their action via endosomal PTHR/GS/cAMP signaling and are thought to favor bone-resorption responses associated with sustained calcium release; conversely, RG selective ligands (such as PTHrP) induce short and transient action that originate receptors localized at the plasma membrane and are believed to favor bone anabolism responses. Live cell microscopy methods described here coupled to those previously reported (Vilardaga et al., 2013) are critical to advance the new signaling model of PTHR that is illustrated in Figure 1. Mass spectrometry-based endosomal PTHR proteomics is powerful in revealing the molecular mechanisms of prolonged PTHR endosomal signaling. A number of interesting proteins were identified in the endosome PTHR signaling complexes, including the PTHR, a set of G protein bγ subunits (Gbγ), several GAPs (GTPase-activating proteins) and GEFs (guanine nucleotide exchange factors), PP2A, and β-arrestins. Further investigation of these PTHR interacting proteins may shed light on the molecular mechanisms of prolonged endosomal cAMP production in GPCR signaling.
This study was supported by the National Institutes of Health (NIH) under Award numbers R01 DK087688 (JPV) and R01 DK102495 (JPV).