To determine whether the D-AKAP2 Ile Val variation resulted in an alteration in its binding properties to PKA, the binding of each variant to the regulatory subunits of PKA was investigated. Binding of each AKB domain variant to PKA was first examined by using an in vitro pull-down assay, in which the 40 C-terminal residues of D-AKAP2 containing the AKB were fused to glutatione S- e/\ transferase (GST) (Fig. 2b). The Ile Val substitution resulted in an aterla i isoform-specific difference in PKA binding. The RI isoform of PKA bound with a significantly higher affinity to the Val variant (Fig. 2b). The higher affinity was seen for both the mouse and the human AKB domains. The residue differences between mouse and ate/\ human downstream of the Ile Val position had no effect on the binding properties. Moreover, there was no difference in binding of JEN  the variants to the RII isoform (Fig. 2b).

Fig.3. AssociationoftheAKBdomainofD-AKAP2withRI(cid:7)andRII(cid:7)in10T(1(cid:1)2) cells. Through fusion with a mitochondrial targeting domain, the AKB domain directsdifferentamountsofRIandRIItothemitochondria.TheValvariant(V24) effectively binds RI(cid:7)(a–c) and targets RI(cid:7)-D(cid:1)D-GFP to mitochondria (b). The Ile variant(I24)bindstoRI(cid:7)lesseffectively(d–f)andappearstobedistributedevenly in the cells (e). In contrast, both variants show no apparent differences for (g–l) binding; they all colocalize at mitochondria (h and k).

Val variant is the apparent deleterious allele in a European- American cohort, and this finding is replicated in Hispanic Americans. The variant maps to the conserved AKB domain of the D-AKAP2 gene. The I646V variation impacts the binding to PKA in an isoform-specific manner both in vitro and in cells. The rr @  Ile variant binds three-fold weaker to the RI isoform than to the Val variant. At the cellular level, this affinity difference ati results in a dramatic decrease in compartmentalization of RI for the Ile variant. In the accompanying manuscript by Burns- vueie  Hamuro et al. (29), we provide further evidence that the Ile Val nela i variant is critical for binding to only the RI isoform of PKA.

Toassessthemagnitudeoftheaffinitydifference,bindingofboth variants to the R isoforms was analyzed in a quantitative assay. Peptides of each AKB variant were synthesized and fluorescently labeled. Binding of these peptides to the regulatory subunit iso- forms was monitored by an increase in steady state fluorescence anisotropy (Fig. 2c). There was no difference in binding of the AKB i=Ta ¢ Ip i variants to either the RII or RII isoforms. In addition, the RII eeTa i isoforms bound tighter to the variants than the RI isoform (Fig.  2c Inset). However, as indicated by the pull-down experiments, RI displayed differential binding to the AKB variants. The Val variant had a nearly three-fold increase in binding affinity when compared with the Ile variant. The interaction was specific because no binding lat was observed to a deletion construct of RI that lacked the AKAP ARONETa). binding domain (delta1–91RI ).

How the RI specific binding differences of the D-AKAP2 variants translate into a health risk is likely to be complex. Com- partmentalization by AKAPs is known to play an important role in the specificity and regulation of cAMP-mediated signaling of PKA (19). The cellular evidence presented here by using the mitochon- drial-targeted form of D-AKAP2 suggests that the Ile variant has aaa  reduced ability to compartmentalize RI PKA. Therefore, de- creased compartmentalization of type I PKA may have an advan- tageous health effect. This effect could be direct by decreasing PKA signaling through D-AKAP2-Ile anchored PKA or indirect by aTa ii redistributing the local pools of RI in cells that have an important impact on health.

To determine whether the observed in vitro affinity differences resulted in a difference in cellular compartmentalization, we ex- aminedtheassociationoftheAKBvariantsandthePKAregulatory subunits in cells. In this assay, the AKB domains of D-AKAP2 from mouse (Val) and human (Ile) were fused to the mitochondrial anchoring domain of D-AKAP1, respectively, and tethered to the outer mitochondrial membrane (27, 28). The binding of the AKB domain to the PKA regulatory subunits was detected as the cotransfected PKA regulatory domains colocalized to the mito- chondria.Asexpected,boththeValandIleAKBvariantscantarget Ta RII to the mitochondria effectively, in accordance with their ~~ W.lat similar affinity in vitro (Fig. 3 h and k). However, for RI there was a difference in colocalization between the variants. The Val variant effectively targeted RI(cid:7) to the mitochondria (Fig. 3b). The Ile variant, however, was unable to target RI(cid:7) , which was evenly diffused in the cytosol (Fig. 3e) demonstrating that the Ile variant of D-AKAP2 has impaired ability to sequester RI(cid:7) .

Increasing evidence suggests that the RI and RII isoforms of PKA have distinct functions (30). The RI isoform has been implicated in a variety of biological functions such as cell proliferation (31), tumor suppression (32, 33), immune regula- tion (34), and embryonic development (35, 36). In addition, the wae RI isoform plays a significant role in maintaining cAMP regulation of PKA as evidenced by the embryonic lethality of mice deficient in the gene (35). Interestingly, these mice have defects in cardiac morphogenesis.

The correlation of the I646V variant with the EKG PR interval indicates that this polymorphism might be a predisposing factor for a cardiac phenotype. Individuals homozygous for the Val variant exhibit shorter atrium depolarization intervals (PR) than individ- uals homozygous for Ile. This phenotypic correlation combined with literature reports supporting a role for AKAP-mediated PKA signaling in normal cardiac function suggest plausible hints of how the Val variant may lead to pathogenesis.

Discussion