To identify common polymorphisms in the D-AKAP2 gene, we sequenced the 15 exons and 100,200 bp of the flanking sequence (Fig. 4, which is published as supporting information on the PNAS website, www.pnas.org). The analysis of 36 chromosomes revealed only two additional polymorphisms: an A to G SNP in intron 10, six nucleotides downstream from exon 10 (In10), and an A to G SNP in exon 14, leading to an amino acid substitution Ile to Val at position 646 (I646V). Individual genotyping of the European-American samples showed that the intron 10 SNP exhibits no morbidity-association. The I646V polymorphism, however, was found to be significantly different between young and old in both male (P = 0.03) and female (P = 0.009) groups (Table 3). There was no significant difference between young males and females and between old males and females. We applied the Bayesian coalescent theory-based method (20) to construct haplotypes at these three tightly linked sites for each subject. The estimates of the disequilibrium (D) between the 3 UTR and the I646V and In10 SNPs were 0.991 and 0.255 (r2 = 0.55 and 0.03), respectively. The distance (eave) between the markers in strong disequilibrium, 3 UTR and I646V, which both showed association, is 4 kb, whereas the intron 10 SNP is located 23 kb upstream of I646V (Fig. 4).

Table 3 shows a comparison of allele frequencies between young and old groups for each SNP with frequencies for the G allele. At the 3 UTR and the I646V variable sites, GG homozygotes were reduced and AA homozygotes increased in the older sample of both genders. This further supports the hypothesis that the G allele, which determines the Val allele at I646V, is associated with a negative health impact.

The variable position resulting from the SNP (I646V) is located in the AKB domain of D-AKAP2. This domain is the docking site for PKA (13) and is highly conserved among species (Fig. 2a). Similar to other AKAPs, the AKB domain is predicted to form an amphipathic helix with hydrophobic amino acids on one face of the helix (21, 22). The length of the hydrophobic side chains on the hydrophobic face of the helix can alter PKA binding specificity (23, 24). The AKB domain of D-AKAP2 interacts with RI and RII isoforms of PKA and determines the extent of subcellular localization of PKA. The presence of two potential RGS (regulator of G protein signaling) motifs suggests the involvement of D-AKAP2 in G protein-mediated signal transduction (13, 18, 25). A putative PDZ-binding motif (TKL) at the C terminus is a protein-protein recognition module that plays a central role in organizing diverse cell signaling assemblies (26). The R249H variant is located in a spacer separating two halves of a potential RGS domain. The conservation of this position and surrounding sequence is very low compared with the AKB domain. Because there is no interaction partner identified for this spacer, the potential functional effect of R249H was not analyzed further.

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 glutathione S-transferase (GST) (Fig. 2b). The Ile Val substitution resulted in an 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 human downstream of the Ile Val position had no effect on the binding properties. Moreover, there was no difference in binding of the variants to the RII isoform (Fig. 2b).

To assess the magnitude of the affinity difference, binding of both 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 theIn 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 was observed to a deletion construct of RI that lacked the AKAP ARONETa binding domain (delta191RI). How the RI-specific binding differences of the D-AKAP2 variants translate into a health risk is likely to be complex. Compartmentalization 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 mitochondrial-targeted form of D-AKAP2 suggests that the Ile variant has a reduced ability to compartmentalize RI PKA. Therefore, decreased compartmentalization of type I PKA may have an advantageous health effect. This effect could be direct by decreasing PKA signaling through D-AKAP2-Ile anchored PKA or indirect by redistributing the local pools of RI in cells that have an important impact on health.