br Acknowledgments br Introduction Angiotensin II AngII is a

Acknowledgments
Introduction Angiotensin II (AngII) is among the most potent vasoactive substances produced in humans. Its effects are numerous, from vasoconstriction to control of fluid and electrolytes balances. Many of the physiological and pathological effects of AngII are mediated by the angiotensin II receptor type 1A (AT1R). Activation of AT1R triggers cardiovascular remodeling in subjects with hypertension and heart failure [19]. Pharmacological inhibition of AT1R has been exploited extensively in the treatment of hypertension and heart failure. Classically, AT1R is a 7-pass transmembrane receptor associated with Gαq/11, whose dissociation upon AngII binding to AT1R is associated with increases in intracellular free Ca2+ concentration, followed by both pre-genomic actions such as contractions of the heart and muscle cells and genomic actions that alter the expression of various genes [17]. AT1R also interacts with β-arrestin, triggering a G protein-independent cascade of downstream events, with distinct physiological outcomes [11]. Additionally, the activities of AT1R are regulated by receptor-interacting proteins [1], [6], [32]. Nevertheless, aspects of signaling via AT1R at the receptor level and its regulatory inputs remain incompletely understood. Calmodulin is a ubiquitous transducer of intracellular Ca2+ signals. It is estimated to bind up to 300 intracellular proteins [21]. Despite its universal requirement, CaM is not expressed sufficiently for all its Ciprofibrate within the cell [10]. Shortage of CaM relative to its binding sites [12], [14], [23], [24], [31], coupled with disparate binding affinities for its target proteins from ∼10−11 to 10−5 M, has made CaM a target for dynamic competition among its binding proteins, leading to new modes of functional coupling in cells [23], [24]. Over the last two decades, CaM has been shown to interact with several GPCRs, such as the metabotropic glutamate receptors mGluR1 and mGluR5 [18], opioid μ receptors [30], the parathyroid hormone receptor 1 [15], the 5-HT(1A) and 5-HT(2C) receptors [3], [28], the D2 dopamine receptors [7], AT1R [22], [32], and the G protein-coupled estrogen receptor 1 (GPER) [25], [27]. CaM binding to the 5-HT(1A) alters receptor phosphorylation and G protein coupling [28]; while its association with 5-HT(2C) and GPER is important receptor-mediated ERK1/2 activation [3], [25]. In 1999, a CaM-binding domain was identified at the juxtamembranous region of the 4th submembrane domain (SMD4JM) of AT1R (a.a. 305 – 327, rat sequence) [22]. The interaction was shown to be Ca2+-dependent. In 2013, Zhang et al. identified another CaM-binding domain on SMD3 (a.a. 215 – 232) and confirmed the previously identified domain on the juxtamembranous domain of the 4th submembrane domain of the receptor [32]. In that work, CaM was shown to compete for Gβγ association with both the partial SMD3 and SMD4JM peptides in vitro, indicating a role for CaM binding in Gβγ coupling. Despite identification of two CaM-binding domains on AT1R, questions remain to be answered regarding total number of binding sites, biochemical properties and regulatory inputs of the interactions with CaM. Additionally, the functional impact of these interactions in cells are unknown. For example, are segments a.a. 305–327 (rat) in SMD4JM[22] and a.a. 215–232 (rat sequence) in SMD3 [32] the only locations where CaM interacts with AT1R? How much Ca2+ is needed for these interactions to occur? In this regard, for most known CaM-GPCR interactions, information is only available whether CaM binding to GPCR domains occurs in saturating Ca2+ or absence of Ca2+. The lack of EC50(Ca2+) values for CaM-GPCR domain interactions makes it difficult to predict when an interaction occurs in cells. Additionally, what are the functional implications of AT1R-CaM interactions? We recently used a FRET-based method to identify and characterize four distinct CaM-binding domains in the G protein-coupled estrogen receptor 1 (GPER) [27]. The method is highly specific and allows for precise determinations of apparent K values of CaM binding to the GPCR domains as inserts between the donor-acceptor FRET pair. These values serve as sensitive parameters to determine the binding domains, especially those that do not conform to any known binding motifs. Simultaneous measurements of biosensor-CaM interaction and responses of suitable Ca2+ indicator also enables determination of the precise EC50(Ca2+) values of the interactions. These values, coupled with measured free Ca2+ concentrations in cells, enable predictions of the physiological scenarios in which a particular CaM–GPCR interaction may occur [27].