At the protein level Cα1 and Cα2 are 97% homologous and only differ at the N-terminal end. Based on this we investigated to what extent differences at the N-terminus may influence splice variant-specific activities that may have biological importance. We found that Cα2 expressed in bacteria was not captured by inclusion bodies as was the case with Cα1. Moreover, the specific activity of Cα2 was lower compared to Cα1. Apart from these differences we observed that Cα2 was highly similar to Cα1 in all parameters measured. This included association of Cα2 with RI and RII to form cAMP-sensitive holoenzymes both in vivo and in vitro. Furthermore, Km values of Cα2 for Kemptide and ATP were comparable to those determined for Cα1. This was also the case for the ability of RI, RII and PKI to inhibit Cα2 enzyme activity in vitro (data not shown). Finally, KD values as measured by SPR were shown to be comparable between Cα1 and Cα2 towards the RI and RII subunits as well as PKI.
Several reports imply that N-terminal modifications of Cα1 introduce specific features that may have biological consequences. To this end it has been suggested that phosphorylation of Ser10 in Cα1 introduces an electrostatic force which may help the C subunit to remain soluble even when myristylated [55;56]. Moreover, two reports have demonstrated that the N-terminal myristyl moiety of Cα1 is embedded in a hydrophobic pocket encompassed in the large lobe [57;58]. Mutation of Gly1 to Ala rendering the Cα1 non-myristylated, demonstrated that myristylation was non-essential for conformation and enzyme activation, and was not required for Cα1 interaction with other proteins including various substrates and the R subunits [21
]. The fact that Cα2 is not myristylated and displays comparable activities with myristylated Cα1 suggests that myristylation is not essential for catalytic activity, holoenzyme formation and inhibition by PKI. This is further supported in that deletion of the entire Cα1 N-terminus did not severely interfere with catalytic activity and inhibitor binding despite that deletion caused thermo instability [25
]. This also suggests that the amino acids 2 (Asn) and 10 (Ser) of Cα1 are not essential for activity a suggestion which is supported by our results on Cα2.
Cα1 and Cβ1 which are 100% identical at the N-terminus, but only 91% identical in the sequence encode by exon 2 through 10, have different apparent sizes (40 and 41 kDa) and possess distinct biochemical properties both in vitro and in vivo [60; 31]. These differences include differential Km values for certain peptide substrates and that Cα1 but not Cβ1 is inhibited by substrate concentrations above 100 μM. In addition, they display distinct IC50 values for PKI and RIIα. Taken together with our results this may suggest that the amino acid sequence encoded by exon 2 through 10 and not exon 1 influence C subunit activities such as holoenzyme formation, enzyme activity and inhibition by R and PKI. This may further imply that Cα1 and Cβ1 have distinct roles in regulating cellular processes. This was recently shown in T cells which express Cα1, Cβ1 and Cβ2. In that study Cα1, but none of the Cβ forms, mediated the inhibitory effect of cAMP on immune cell reactivity in vivo [17;61]. In light of these observations it is also of interest to note that Cα2 but not Cα1 is required for sperm cell forward velocity and male fertility, despite 100% identity at the amino acid sequence encoded by exon 2 through 10 [20;62]. However, since Cα2 is the sole C subunit in sperm cells [9;10;63], the difference observed may only be ascribed to tissue-specific expression and not sequence-specific differences.
In contrast to Cα1 it is expected that the hydrophobic pocket in which the myristyl group is submersed in Cα1, is constitutively empty and exposed to the surroundings at all time in non-myristylated Cα2. It has been speculated whether exposure of the hydrophobic pocket would introduce more lipophilic properties to the Cα2 subunit [64
]. Support for such a hypothesis is found in a by a study demonstrating that binding of Cα1 to RII induced a unique conformation that is associated with exposure of the hydrophobic pocket to the surroundings due to increase in N-terminal flexibility of the N-myristate away from the large lobe. This renders Cα1 more hydrophobic and promotes membrane association of the PKA II holoenzyme only [64
]. Therefore it may be suggested that exposure of the hydrophobic pocket serves features such as isoform specific features and subcellular localization of the C subunit. To this end it is interesting to note that Cα2 is associated with the sperm tail in the presence of detergent treatment with 1% Triton X-100 .and, after a challenge with 2 mM cAMP [10
]. This may be indicative of a direct association of Cα2 with subcellular structures. To what extent such attachment involves the hydrophobic pocket remains unknown. In other cells and tissues, C subunits targeted to subcellular structures independent of the R subunit and traditional A-kinase anchoring proteins have been demonstrated. To day a number of C subunit binding proteins have been identified. These include PKI, A-kinase interacting protein 1 (AKIP1), homologous to AKAP95 (HA95), inhibitor of NFkappaB kinase (IκB), Caveolin-1 and p75 neutrophine receptor (p75NTR) [65
]. To what extent Cα2 is targeted to the sperm cell midpiece through a C interaction protein and if specificity of binding is retained in the hyper variable N-terminal end remains to be shown. However, it should be noted that deamination of the Asn2 moiety in Cα1 helps fine-tuning enzyme distribution within the cell in vivo
]. Moreover, p75NTR was shown to specifically bind to the Cβ splice variant Cβ4ab [69
], which is encoded with unique N-terminal domain that may not undergo the same posttranslational modifications as Cα1 [15;69]. Together this may imply that the N-terminal end may be important for targeting and specificity of subcellular localization of the various C subunits.