The Open Question of How Gpcrs Interact with GPCR Kinases (Grks)
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biomolecules Article The Open Question of How GPCRs Interact with GPCR Kinases (GRKs) M. Claire Cato 1 , Yu-Chen Yen 2 , Charnelle J. Francis 3, Kaely E. Elkins 3, Afzaal Shareef 4 , Rachel Sterne-Marr 3 and John J. G. Tesmer 2,* 1 Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA; [email protected] 2 Departments of Biological Sciences and Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA; [email protected] 3 Biology Department, Siena College, Loudonville, NY 12211, USA; [email protected] (C.J.F.); [email protected] (K.E.E.); [email protected] (R.S.-M.) 4 Department of Chemistry and Biochemistry, Siena College, Loudonville, NY 12211, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-765-494-1807 Abstract: G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic pro- cesses, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR–GRK Citation: Cato, M.C.; Yen, Y.-C.; complexes would help refine these models but are, so far, lacking. Here, we assess three distinct Francis, C.J.; Elkins, K.E.; Shareef, A.; models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to Sterne-Marr, R.; Tesmer, J.J.G. The generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested Open Question of How GPCRs Interact with GPCR Kinases (GRKs). by the two newest models. Biomolecules 2021, 11, 447. https:// doi.org/10.3390/biom11030447 Keywords: G protein-coupled receptor; G protein-coupled receptor kinase; protein structure; phos- phorylation; complexes; allostery; models Academic Editor: Karsten Melcher Received: 19 February 2021 Accepted: 15 March 2021 1. Introduction Published: 17 March 2021 G protein-coupled receptors (GPCRs) are the largest and the most diverse receptor family found in eukaryotes and are involved in regulating many diverse physiological pro- Publisher’s Note: MDPI stays neutral cesses. Upon activation by extracellular stimuli such as hormones and neurotransmitters, with regard to jurisdictional claims in GPCRs transmit signals across the membrane to trigger intracellular signaling cascades. published maps and institutional affil- The hallmark seven-transmembrane topology of GPCRs was proposed in the 1980s based iations. on the primary structure of bovine rhodopsin and the β-adrenergic receptor [1–4]. To date, more than 800 GPCR sequences have been identified in the human genome. Based on the sequence and functional similarity, the GPCR superfamily is divided into six classes, of which the rhodopsin-like family (group A) is the largest [5,6]. Copyright: © 2021 by the authors. In cells, unliganded GPCRs exist in an equilibrium between inactive and active states, Licensee MDPI, Basel, Switzerland. with inactive states being the most prevalent. Upon agonist binding, the equilibrium This article is an open access article shifts toward active conformations that couple with downstream transducers such as het- distributed under the terms and erotrimeric G proteins, GPCR kinases (GRKs), and arrestins. Heterotrimeric G-proteins are conditions of the Creative Commons composed of Gα,Gβ, and Gγ subunits, with Gβ and Gγ forming a constitutive heterodimer Attribution (CC BY) license (https:// (Gβγ). The activation of GPCRs induces conformational changes on the Gα subunit that creativecommons.org/licenses/by/ α 4.0/). allows the exchange of bound GDP for GTP. As a result, the G subunit dissociates from Biomolecules 2021, 11, 447. https://doi.org/10.3390/biom11030447 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 447 2 of 19 Gβγ subunits, and then the free Gα and Gβγ subunits regulate the activity of downstream effectors such as adenylyl cyclases, phospholipases, ion channels, and Rho guanine nu- cleotide exchange factors, thereby modulating diverse signaling pathways [7]. Meanwhile, GRKs phosphorylate the activated GPCRs, promoting the recruitment of arrestins to form GPCR-arrestin complexes, many of which interact with clathrin-coated pits for endocytosis and receptor internalization [8], and, ultimately, receptor downregulation [9]. Beyond their function in terminating G protein-mediated signaling from GPCRs, the binding of arrestins induces alternative G protein-independent signaling pathways, including the activation of ERK and Src family kinases [10]. The first atomic resolution GPCR structure determined was that of bovine rhodopsin bound to its intrinsic inverse agonist 11 cis-retinal [11]. Seven years later, the structure of the β2-adrenergic receptor (β2AR) in a complex with a diffusible inverse agonist was reported [12]. Both structures represented inactive, relatively stable conformations that revealed in atomic detail the basic core architecture for all GPCRs, wherein the extracellular N-terminus is followed by the characteristic seven transmembrane (TM) helices that are connected by three intracellular loops (ICLs) and three extracellular loops (ECLs). These elements are followed by a short amphipathic helix (H8) that packs against the inner leaflet of the plasma membrane and is often palmitoylated. Finally, the cytoplasmic C-terminus is typically an extended tail that contains multiple serine and threonine residues, some of which are phosphorylated by GRKs upon receptor activation. In some GPCRs, an extended third ICL (ICL3) can harbor the phosphorylation sites required for arrestin recruitment. The activated conformation of GPCRs is relatively unstable and difficult to isolate on its own. Therefore, many GPCR structures that have been resolved in an active state have employed protein complexes that recognize this conformation of the GPCR, such as G proteins or nanobodies [13–16]. The characteristic feature of an activated GPCR is a twisting movement of the cytoplasmic ends of TM5 and TM6 outward from the core of the TM bundle, forming a cytoplasmic cleft defined by residues in TM3, TM5, TM6, and the N-terminus of H8. This pocket accommodates the C-terminus of Gα subunits during G protein coupling. Recently determined structures reveal that arrestins also use a similar readout mechanism, but with an internal “finger loop” occupying the cytoplasmic pocket [17–21]. This provides a simple competitive mechanism for how arrestins block the access of G proteins to active receptors. However, arrestins also bind tightly to the phosphorylated C-tails of these receptors, and thus one arrestin molecule could, in principle, bind at one or the other or both sites, depending on the receptor structure, the extent of phosphorylation, and the phosphorylation pattern itself [22,23]. Ever since GRKs were identified as key regulators of active GPCRs, investigators have sought to determine which structural features on GRKs are used to discriminate between the agonist-free and agonist-bound states of GPCRs and how binding to agonist-bound GPCRs enhances the catalytic activity of the GRK [24–28]. It was also established early on that activated GPCRs are allosteric activators of GRKs, based in part upon the observation that activated receptors with no phosphosites can increase the GRK catalytic efficiency to- wards soluble peptide substrates [28,29], consistent with stabilization of a more catalytically competent state of the enzyme. However, despite a wealth of molecular information from high-resolution crystal structures of five of the seven human GRKs [30–34], the mechanism by which the kinase domain transitions from an inactive to an active state has remained elusive. This is because the N-terminus and active site tether (AST) regions of GRKs, which have long been postulated to play a role in the allosteric activation of GRKs, are typically disordered or adopt inconsistent conformations among the available structures, in part due to interactions mediated by crystal contacts. This behavior also reflects the fact that, with a few notable exceptions, the kinase domains in these structures do not adopt a fully closed conformation, at least based on a comparison to the transition state complex of protein kinase A (PKA) [35], a closely related and well-characterized member of the AGC kinase family to which GRKs belong (Figure1A). The exceptions are a structure of GRK6 bound to an adenosine analog sangivamycin [36] and the recently reported structure of Biomolecules 2021, 11, 447 3 of 19 GRK5 in complex with Ca2+·calmodulin (Ca2+·CaM) [37]. In these models, the kinase domain, N-terminal helix, and AST adopt similar configurations (Figure1B,C). In the first case, the assembly seems to be stabilized by interactions between the N-terminal helix with a fortuitous crystal contact and, in the latter case, by Ca2+·CaM. Thus, whatever the mechanism of GPCR-mediated GRK activation, it seems