Abstract
The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca(2+) binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca(2+) binding to sites III and IV, and we present a model showing that this could increase Ca(2+) binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ motif (amino acids 39-62), and an adjacent acidic cluster of amino acids (amino acids 28-40). A synthetic peptide spanning residues 28-62 faithfully mimics intact PEP-19 with respect to increasing the rates of Ca(2+) association and dissociation, as well as binding preferentially to the C-domain of CaM. In contrast, a peptide encoding only the core IQ motif does not modulate Ca(2+) binding, and binds to multiple sites on CaM. A peptide that includes only the acidic region does not bind to CaM. These results show that PEP-19 has a novel acidic/IQ CaM regulatory motif in which the IQ sequence provides a targeting function that allows binding of PEP-19 to CaM, whereas the acidic residues modify the nature of this interaction, and are essential for modulating Ca(2+) binding to the C-domain of CaM.
Highlights
PEP-19 (Purkinje cell protein 4 or pcp4) and RC3 are small proteins expressed primarily in neuronal tissues, but with no known activity other than binding to CaM in the presence or absence of Ca2ϩ
We show here that the consensus IQ CaM binding motif is necessary, but not sufficient to mimic the effect of intact PEP-19 on CaM
The corresponding sequences from myosin V and the voltage-gated Ca2ϩ channel Cav1.2 are shown in Fig. 1A to emphasize the absence of acidic clusters in these IQ motif proteins
Summary
PEP-19 (Purkinje cell protein 4 or pcp4) and RC3 (neurogranin or Ng) are small proteins expressed primarily in neuronal tissues, but with no known activity other than binding to CaM in the presence or absence of Ca2ϩ. RC3 accelerates the rate of Ca2ϩ dissociation from CaM, but has a lesser effect on the association rate, thereby decreasing the affinity of binding Ca2ϩ to the C-domain of CaM [6] Both PEP-19 and RC3 exert these effects even when CaM is bound to CaM-dependent protein kinase II (CKII␣) [5, 6]. These results suggest that PEP-19 and RC3 could have broad extrinsic effects on CaM-related signaling pathways by modulating the Ca2ϩ binding properties of free or enzyme-bound CaM This is consistent with the phenotype of RC3 knock-out mice, which show defects in synaptic plasticity [7], attenuated phosphorylation of hippocampal protein kinase A and C substrates [8], and altered Ca2ϩ dynamics in cortical neurons [9]. We propose that this acidic/IQ sequence constitutes a new CaM regulatory motif
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