How calmodulin interacts with the adenosine A(2A) and the dopamine D(2) receptors

Abstract

Receptor heteromerization is a mechanism used by G protein-coupled receptors to diversify their properties and function. We previously demonstrated that these interactions occur through salt bridge formation between epitopes of the involved receptors. Recent studies claim that calmodulin (CaM) binds to an Arg-rich epitope located in the amino-terminus of the dopamine D(2) receptor third intracellular loop. This is the same epitope involved in adenosine A(2A)-D(2) receptor heteromerization, through Coulombic interaction between the Arg residues and a phosphorylated serine (pS) located in the medial segment of the C-terminus of the A(2A) receptor. Mass spectrometric analysis indicates that an electrostatic interaction involving the D(2) receptor Arg-rich epitope and several CaM acidic epitopes are mainly responsible for the D(2) receptor-CaM binding. CaM could also form multiple noncovalent complexes by means of electrostatic interactions with an epitope localized in the proximal segment of the C-terminus of the A(2A) receptor. Ca(2+) disrupted the binding of CaM to the D(2) but not to the A(2A) receptor epitope, and CaM disrupted the electrostatic interactions between the D(2) receptor epitope and the more distal A(2A) receptor epitope. A model is introduced with the possible functional implications of A(2A)-D(2)-CaM interactions. These in vitro findings imply a possible regulatory role for CaM in receptor heteromers formation.

Figure 1

(a) Bovine CaM sequence and (b) bovine CaM model.

Figure 2

Binding of the D₂ NI3L and the A_2A−1 epitopes to CaM. Spectra of (a) NCX of CaM and the D₂ NI3L epitope (VLRRRRKRVN). (b) No NCX form between CaM and the modified D₂ NI3L epitope (VLAAAKAVN) where Arg were replaced by Ala. (c) Disruption of the interaction of the D₂ NI3L epitope (VLRRRRKRVN) and the A_2A−2 epitope (SAQEpSQGNT) by CaM. Although both peptides are present, no NCX forms between these epitopes in the presence of CaM. (d) Mixture of CaM and the A_2A−1 epitope (RIREFRQTFR).

Figure 3

Fragments generated by the chymotryptic digest of CaM, the NCX of CaM with the D₂ NI3L and the A_2A−1 epitopes. Spectra of enzymatic digest of (a) CaM alone, (b) mixture of CaM and the D₂ NI3L epitope (VLRRRRKRVN), and (c) mixture of CaM and the A_2A−1 epitope (RIREFRQTFR). * denotes CaM fragments.

Figure 4

(a) Spectrum of a mixture of CaM and the D₂ NI3L epitope (VLRRRRKRVN) in the presence of CaCl₂ (10 mg/dL), showing a decrease in the relative abundance of NCX of CaM with the D₂ NI3L epitope in the presence of Ca²⁺. (b) Lack of changes in the binding of CaM to the A_2A−1 epitope in the presence of Ca²⁺. Spectra of a mixture of CaM and the A_2A−1 epitope (RIREFRQTFR) in the absence and presence of CaCl₂ (10 mg/dL). (c) Spectra of mixture of the D₂NI3L epitope (VLRRRRKRVN) and A_2A−2 epitope (SAQEpSQGNT) showing no significant change in NCX formation in the absence and presence of Ca²⁺.

Figure 5

Scheme of hypothetical functional interactions between CaM and A_2A and D₂ receptors. (a) In the absence of CaM, A_2A receptor binds to the D₂ receptor by an electrostatic interactions between the A_2A−2 epitope (SAQEpSQGNT), localized in the medium segment of the C-terminus of the A_2A receptor (pink box), and the D₂NI3L epitope (VLRRRRKRVN), localized in the N-terminal segment of the third intracellular loop of the D₂ receptor (blue box). (b) A_2A−D₂ receptor heteromerization could also involve CaM as a linker, by its ability to simultaneously bind to the D₂NI3L epitope and to the A_2A−1 epitope (RIREFRQTFR), localized in the proximal segment of the C-terminus of the A_2A receptor (red box). (c–d) Ca²⁺ (black dots) disrupts the binding of CaM to the D₂ receptor but not to the A_2A receptor, which leaves the D₂NI3L epitope to interact with the A_2A−2 epitope or with G_i/o proteins (see text).