Calmodulin, an antagonist of dopamine receptor signalling

Research project P 14273 Calmodulin, an Antagonist of Doramine Receptor Signaling Christian NANOFF 08.05.2000 The catecholamine dopamine acts as a neuromodulator in the CNS. The receptors for dopa-mine belong to the class of G protein coupled receptors linked in a subtype specific manner (D1 -stimulatory, D2 -inhibitory) to the formation of cAMP. Among them, the D2 -receptor subtype is highly relevant as a drug target in the pharmacotherapy of human diseases (schizophrenia, Parkinson`s disease). Neostriatal neurons are substrate to dopaminergic innervation; when neostriatal neurons fire nerve impulses the cell is exposed to oscillating intracellular Ca2+ due to Ca2+-currents induced by excitatory neurotransmitters. Thus, it is conceivable that D 2 - receptor signaling proceeds in the presence of elevated intracellular Ca2+concentrations. Ca2+- and cAMP- dependent signaling pathways are under mutual influence through the acti-vation of specific protein kinases (PKC, PKA) which are able to tune the signaling efficiency in other pathways. In nerve cells, however, we suggest that D2 -signaling is under the acute control of Ca2+ oscillations and that the messenger in this cross-talk is the intracellular Ca2+-binding protein calmodulin (CaM). Calcium activated calmodulin (Ca 2+/CaM) is proposed to bind directly to the D 2 -dopamine receptor; this interaction modulates the ability of the D2 -receptor to activate G proteins and thereby integrates input from Ca2+-signaling pathways to those controlled by the D 2 -receptor. In the current proposal we have included preliminary evidence that the D 2 dopamine receptor binds calmodulin with a domain that is competent in activating Gi (i.e. the aminoterminal part of the 3rd intracellular loop), and which reveals a calmodulin-binding motif. Therefore, calmodulin may represent a biochemical link between Ca2+- dependent signaling and the D2 dopamine receptor. Our grant application proposes three lines of experiments which are intended to evaluate i. the affinity and the structural requirements of Ca2+/CaM binding to the D2 dopamine receptor. ii. if the ability of the receptor to bind calmodulin and to activate G i are integral or can be dissociated through permutation of the receptor peptide sequence. iii. if intracellular Ca2+-oscillations govern the D2 -receptor mediated modulation of ion currents in nerve cells.

The D2 -receptor subtype is highly relevant as a drug target in the pharmacotherapy of human diseases (schizophrenia, Parkinson`s disease). The D2 -dopamine receptor is a predominantly Gi/Go -coupled receptor but accessory proteins may impinge on receptor signaling. We showed that calmodulin, the cellular calcium sensor inhibits receptor-mediated G protein activation. CaM binds to the activation competent domain in the receptor (N- terminal segment of the third intracellular loop) with an affinity of ~100 nM which drops markedly in the absence of Ca2+. To delineate the biological role of the receptor-CaM interaction we attempted to dissociate the dual ability of the receptor to bind CaM and to activate Gi/o. By permutating the receptor sequence critical in CaM-binding and G protein activation we intended to create a receptor that is blind to CaM but positively couples to the cognate G protein. However, in each of the mutant peptide fragments the activating potency dropped whereas the affinity for CaM was conserved even when closely related amino acids were used for substitution. We have exploited the mutant receptor peptides in order to delineate the molecular mechanisms in receptor- mediated G protein activation. The reaction steps leading to the recognition and activation of the cognate G protein require more than one contact with the receptor. The receptor contact sites can be mapped on the membrane- opposed surface of the G protein heterotrimer. Nevertheless it is an unresolved question in the field which of the contacts brings about the activation switch (the release of pre-bound GDP from the guanine nucletide binding pocket). Our preliminary findings clearly point to the N-terminal domain of the a-subunit as a key domain (about 34 residues out of 330). In order to demonstrate that this domain suffices to trigger activation, the effect of the receptor peptide has to be tested on an N-terminally mutated protein. Our next goal is to generate a truncated version of the protein and to evaluate this hypothesis.