Expression of wild-type RIα in mammalian cells revealed a punctate pattern that is RI specific. (A) Transient transfection of control GFP empty vector or GFP-tagged RIα in HeLa cells (top) and Prkar1a^−/− MEFs (bottom). (B) Expression of fluorescently tagged R-subunit isoforms in HeLa cells (top) and Prkar1a^−/− MEFs (bottom). (C) Coexpression of control GFP empty vector or GFP-tagged RIα with mCherry-tagged PKAc in Prkar1a^−/− MEFs without treatment (left) and with 20 µM forskolin/200 µM IBMX treatment (right). Labels in the top left corner of each image indicate which protein is observed. (D) Representative frames from Video 1 showing Prkar1a^−/− MEFs transiently transfected with GFP-tagged RIα (green) and mCherry-tagged PKAc (red). 10 µM H89 was added to inhibit PKAc activity (last representative frame). (E) Representative frames from Video 3 showing Prkar1a^−/− MEFs transiently transfected with GFP-tagged RIα (green), mCherry-tagged PKAc (red), and CFP-tagged PKI (blue). (D and E) Baseline represents the cell before any treatment, whereas postwash represents the cell after removal of treatment. 20 µM forskolin/200 µM IBMX was added to activate PKAc. DIC, differential interference contrast. wt, wild type. FSK, forskolin. Bars, 20 µm.

Disassociated RIα localized to membranous organelles, and some organelles were ruled out from costaining with organelle markers. (A) Prkar1a^+/+ MEFs were treated with 20 µM forskolin (FSK)/200 µM IBMX and fractionated. Cytosol refers to the cytosolic fraction, mem refers to the membrane fraction, nuc refers to the nuclear fraction, and cytosk refers to the cytoskeletal fraction. (B, top) HeLa cells were transiently cotransfected with GFP-tagged RIα (green) and mCherry-tagged LC3 (red). (bottom) Autophagy was induced in Prkar1a^−/− MEFs, which were transiently transfected with GFP-tagged RIα (green), and then autophagosomes (LC3) were stained (red). (C) Cells were transfected with GFP-tagged RIα (green). Prkar1a^−/− MEFs were stained for lysosomes (LysoTracker), mitochondria (AIF), and peroxisomes (PMP70), whereas HeLa cells were stained for early endosomes (EEA1). (D) Rhodamine-transferrin (Tfn) was used to follow the recycling pathway. (E) Texas red–EGF (EGF) was used to follow the degradative pathway, and cells were fixed at three different time points. EEA1 was stained so that a merge with EGF indicated early endosomes. Bars, 20 µm.

Ultrastructural analysis revealed localization of RIα-GFP in MVBs. Light and electron microscopy pictures were correlated to look for the localization of GFP-tagged RIα. (A and B, top) White/black arrows point to an MVB that was correlated. (bottom) The MVB is indicated by black squares. (bottom right) RIα-GFP is the dark black area within the MVB indicated by a black arrow. (A) Prkar1a^−/− MEFs were transfected with RIα-GFP (green). (B) Prkar1a^−/− MEFs were cotransfected with RIα-GFP (green) and PKAc-mCherry (red) and treated with 20 µM forskolin/200 µM IBMX. (C) Untransfected Prkar1a^−/− MEFs were treated with 20 µM forskolin/200 µM IBMX. An enlarged image of MVBs from within the black square is shown on the right. The MVBs are indicated by black arrowheads.

Disruption of AKAP binding removed the punctate pattern. (A) Control GFP empty vector, GFP-tagged wild-type (wt) RIα, and GFP-tagged deletion mutants, RIα(1–93) and RIα(93–381), were expressed in Prkar1a^−/− MEFs. White arrows indicate a few of the faint puncta that were seen with the expression of RIα(1–93)-GFP. (B) A mutation that has been reported to disrupt D-AKAP1 binding, RIα(I27A), was GFP-tagged and expressed in Prkar1a^−/− MEFs. GFP empty vector and GFP-tagged wild-type RIα were included as controls. (C) mCherry empty vector or mCherry-tagged AKB domain from D-AKAP2 (RI/RII selective, RI selective, or RII selective) was coexpressed with GFP-tagged RIα in Prkar1a^−/− MEFs. Bars, 20 µm.

AKAP11 binds to free RIα and the RIα holoenzyme. (A) Endogenous AKAP11 was immunoprecipitated from HEK 293 cells in the absence and presence of 20 µM forskolin (FSK)/200 µM IBMX. Samples from the lysate and from the elution of the immunoprecipitation (IP) were immunoblotted (IB) with antibodies against RIα, PKAc, or AKAP11. (B) HEK 293 cells were transiently transfected with control FLAG empty vector, FLAG-tagged RIα, or FLAG-tagged RIα(R211K), an RIα mutant that is defective in cAMP binding. Samples from the lysate and from the elution of the IP were immunoblotted with antibodies against AKAP11, PKAc, or FLAG. (A and B) The lanes marked Lysate were loaded with 5% of the total cell lysates. (C) HeLa cells and Prkar1a^−/− MEFs were transiently transfected with GFP-tagged RIα (green) and stained for endogenous AKAP11 (red). Bars, 20 µm.

AKAP11 targets free RIα. (A) Prkar1a^−/− MEFs were transiently cotransfected with GFP-tagged RIα (green) and mCherry-tagged PKAc (red) and stained for endogenous AKAP11 (blue). (B) Prkar1a^−/− MEFs were transiently cotransfected with GFP-tagged RIα (green) and mCherry-tagged PKAc (red), treated with 20 µM forskolin (FSK)/200 µM IBMX, and then stained for endogenous AKAP11 (blue). (C) siRNA of AKAP11 effectively knocked down the levels of endogenous AKAP11 protein in Prkar1a^−/− MEFs after 1–2 d. No effect on the expression level of transiently transfected RIα-GFP was observed, as judged by immunoblotting of whole-cell extracts with antibodies against AKAP11, RIα, Hsp90, or tubulin. (D) Knockdown of endogenous AKAP11 disrupted the formation of RIα-GFP puncta in Prkar1a^−/− MEFs. Ctrl, control. wt, wild type. Bars, 20 µm.

Free RIα is recruited to MVBs by AKAP11. A model for the mechanism of targeting RIα is depicted. AKAP11 binds to the RIα holoenzyme. Upon activation of PKA, AKAP11 stays bound to RIα, which is recruited to multivesicular bodies (MVBs). If the RIα holoenzyme is not activated, as in the case of RIα(R211K):PKAc, RIα does not get targeted. Likewise, free RIα is not targeted properly when AKAP11 binding is disrupted, as in the case with the RIα(I27A) mutation or in the presence of RI-selective AKAP disruptors. R, RIa; C, PKAc; wt, wild type.