Oocyte experiments

Stage V or VI Xenopus oocytes were prepared in Julius-von-Sachs Institute, Würzburg. After the injection of cRNAs, oocytes were maintained in the dark for 3 days at 18°C in ND96 solution: 96 mM NaCl, 2mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 50 μg/ml gentamycin pH 7.4. The amount of cRNA injected per oocyte in Figs. ​Figs.1A;1A; ​A;2A,2A, D, and E; and ​and4A–B4A–B was adjusted to keep copy number approximately equal: bPAC, Lyn-bPAC, and 2xLyn-Myc-bPAC (20 ng); Glyco-HA-bPAC (25 ng); Venus or eYFP tagged soluble bPAC variants, Lyn-Venus-bPAC, and 2xLyn-Venus-bPAC (30 ng); and CD8-Venus-bPAC and Glyco-Venus-bPAC (35 ng). For the remaining PACs, the cRNA injection quantity is 30 ng/oocyte unless otherwise indicated.

Oocytes were either kept in the dark or illuminated for 30 s with 473 nm blue light at 0.3 mW mm^-2 before homogenization. Under far-red light, five oocytes were pooled and homogenized by pipetting in 0.1 N HCl. Cell debris was removed by 10 min 30,000g centrifugation at room temperature. The supernatant was collected and cAMP was quantified using the DetectX High Sensitivity Direct Cyclic AMP Chemiluminescent Immunoassay Kit (Arbor Assays).

Fifteen control oocytes (without cRNA injection) or 15 oocytes pre-injected with cRNA encoding soluble bPAC variants were pooled and washed 3 times in ice-cold solution A (75 mM Tris-HCl, 100 mM NaCl, 2 mM MgCl₂, 5 mM DTT, 1x Protease Inhibitor Cocktail (Roche), pH 7.4). After gentle homogenization by pipetting in 450 μl ice-cold solution A, the yolk and cellular debris were sedimented by 30,000×g centrifugation at 4°C for 20 min. The lipid layer on the surface of the supernatant was removed, and the supernatant transferred to a new tube and centrifuged at 30,000×g at 4°C for another 20 min to remove the residual lipid layer. The oocyte soluble extract (supernatant) was then ultrafiltrated by centrifugation with the Amicon Ultra-0.5 Centrifugal Filter Unit. Small proteins, endogenous ATP/GTP, cAMP/cGMP, and other small molecules were removed by the ultrafiltration step. The crude soluble extracts were then ready for the in vitro reactions. This preparation process was performed under red light.

To determine the endogenous PDE activity, 0.15 μM cAMP was added to the crude soluble extracts with or without IBMX. To determine either endogenous or soluble PAC-induced adenylyl cyclase activity, reactions were started by adding ATP (final concentration 1 mM) to the crude soluble extracts in the dark (under far-red light) or blue light as indicated in the figure legends.

Aliquots were taken from the in vitro reaction mix and stopped by adding 19 volumes of Sample Diluent (containing 0.1 N HCl, Arbor Assays) at the indicated time points (in Fig. ​Fig.1B1B and C). For reaction with the soluble PAC expressing crude extracts, the aliquots were taken and stopped at 3 different time points in the light (1, 4, and 7 min) and in the dark (5, 25, and 45 min). cAMP was quantified using the DetectX High Sensitivity Direct Cyclic AMP Chemiluminescent Immunoassay Kit (Arbor Assays) and turnover calculated after protein quantification (as below).

Fifteen oocytes (uninjected controls or expressing membrane-targeted PACs) were pooled and washed 3 times in ice-cold solution A (see above). After gently homogenizing in 450 μl solution A by pipetting on ice, the yolk and cellular debris were sedimented by 500×g centrifugation at 4°C for 15 min. The supernatant was transferred to a new tube and centrifuged at 30,000×g at 4°C for 20 min for separation of membrane pellets and soluble fraction. The supernatant was discarded. The membrane pellets were gently washed twice with 500 μl solution A and resuspended in solution A at a ratio of 1 oocyte to 4 μl. The resuspended fraction was again centrifuged 500×g at 4°C for 5 min to remove any residual large cellular debris. The supernatant (membrane extract suspension) was used for the in vitro reaction. To start the enzymatic reaction, 4 μl membrane extract was mixed with 36 μl reaction buffer (75 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM DTT, 5 mM MgCl₂, 1 mM ATP, 0.2 mM GTP) either in black Eppi tubes (the dark condition) or under illumination (473 nm, 0.3 mW mm^-2) from the top of the Eppi tube. Aliquots were taken from the in vitro reaction mix and stopped as the soluble protein assay. cAMP concentration was quantified using the DetectX High Sensitivity Direct Cyclic AMP Chemiluminescent Immunoassay Kit (Arbor Assays). The enzyme turnover (μM cAMP/μM enzyme/minutes from start of reaction) was calculated after the fluorescence-based protein quantification (below).

The fluorescence emission of purified Venus and eYFP protein (Evrogen JSC; 1 mg/ml) of known concentration (dilution to 0, 5, 10, 20, 40, 80, and 160 ng with solution A, three repeats) was measured at 538 nm by using a Fluoroskan Ascent microplate fluorometer with 485 nm excitation and used to plot standard curves. The protein levels in Xenopus oocyte membranes and soluble extracts were obtained by measuring fluorescence of the samples on the same fluorometer and comparing to the standard curves.

Venus fluorescence imaging of oocytes was done using a confocal laser scanning microscope (LSM 5 Pascal, Carl Zeiss) equipped with a Zeiss Plan-Neofluar 10× 0.5 nA objective. Images were processed using LSM 5 Image Browser and exported for insertion into figures.