The minor binding pocket: a major player in 7TM receptor activation Mette M. Rosenkilde, Tau Benned-Jensen, Thomas M. Frimurer and Thue W. Schwartz Laboratory for Molecular Pharmacology, Institute for Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark From the deep part of the main ligand-binding crevice, a minor, often shallower pocket extends between the extracellular ends of transmembrane domains (TM)-I, II, III and VII of 7TM receptors. This minor binding pocket is defined by a highly conserved kink in TM-II that is induced by a proline residue located in one of two adjacent positions. Here we argue that this minor bind- ing pocket is important for receptor activation. Function- al coupling of the receptors seems to be mediated through the hydrogen bond network located between the intracellular segments of these TMs, with the allo- steric interface between TM-II and TM-VII being of par- ticular significance. Importantly, the minor binding pocket, especially the proline-kink in TM-II, is involved in G protein versus arrestin pathway-biased signaling, for example in the angiotensin AT1 system. Consequent- ly, this pocket could be specifically targeted in the de- velopment of functionally biased drugs. Introduction The main function of seven-transmembrane (7TM; G pro- tein-coupled) receptors is to convey a broad array of extra- cellular signals across the membrane that in turn control a series of different intracellular signaling pathways [1–3]. Knowledge about this complex and highly diverse allosteric mechanism has increased tremendously over the last couple of years, mainly through the characterization of a number of high-resolution X-ray structures and through novel biophys- ical studies using, for example, electron paramagnetic reso- nance and NMR spectroscopy [4–16]. With respect to ligand binding, most of these studies have focused on the relatively deep part of the so-called main ligand-binding pocket or crevice that is located between the extracellular segments of TM-III, -IV, -V, -VI and -VII (Figure 1) [17]. We now know from the X-ray structures that the prototype monoamine antagonists and inverse agonists bind in this pocket be- tween the AspIII:08/3.32 anchor point and secondary inter- action sites, mainly in TM-V and -VI [7–9,11] (for generic numbering of 7TM residues see Figure 1a and Box 1). This is also where many small-molecule agonists are expected to bind, but probably in a more induced fit manner, where they reportedly stabilize a conformation in which the top of TM- VI tilts inward in the pocket [17,18]. These types of ligands have drawn attention away from the often shallower part of the main ligand-binding pocket located between the extracellular segments of TM-I, -II, -III, and -VII, which is called the minor binding pocket (Figure 1) [17]. However, this pocket is highly important, not only for ligand binding in many receptors, but also for generation of the signal transduction process itself. For example, activation of rhodopsin (after the light-induced isomerization of 11-cis-retinal) actually starts with depro- tonation of the Schiff-base nitrogen of LysVII:10/7.43 (Lys 296 ), which is located in the center of the minor binding pocket on the other side of TM-VII (Figure 1a) [12,19]. In this article, we examine the special structural and func- tional features of the minor ligand-binding pocket, includ- ing the characteristic, highly conserved proline kink in TM- II and the close functional connection to the intracellular signaling mechanism, which is conceivably mediated through the important hydrogen bond network located immediately below the minor binding pocket between the intracellular segments of the TM helices. Ligand binding in the minor pocket In chemokine receptors, most small-molecule agonists and antagonists have extensive interactions in the minor bind- ing pocket because they generally extend into both pockets from their central anchor point, GluVII:06/7.39 (Figure 1a) [20]. For example, TrpII:20/2.60 is known to be important for small-molecule ligands in CC chemokine receptors [21,22]. Position VII:10/7.43, which is located in the center of the minor binding pocket, pointing towards TM-I and TM-II, is also a site frequently used for agonist binding (Figure 1a). In the case of rhodopsin, LysVII:10 (Lys 296 ) is the classical, covalent attachment point for retinal. A number of studies indicate that the corresponding His- VII:10 is a key interaction site for the endogenous agonist, for example, in the adenosine receptor [23,24]. Notably, in the X-ray structure of the adenosine A 2A receptor [14], which is a complex with the ZM241385 antagonist; His- VII:10 forms hydrogen bonds to a group of water molecules, which seem to mimic the missing sugar moiety of a bound agonist (Figure 1d,e). In the chemokine CXCR3 receptor, the corresponding PheVII:10 serves as the last ‘tooth in an aromatic zipper’, which is essential for the function of small allosteric agonists designed to bind in an anchoring metal- ion binding site [25]. Importantly, in the GPR109A recep- tor, the small agonist nicotinic acid binds entirely in the minor ligand-binding pocket, where it is anchored through a charge–charge interaction to ArgIII:12/3.26 (Arg 111 ) that is predicted to reach towards TM-II to make aromatic Opinion Corresponding authors: Rosenkilde, M.M. (rosenkilde@sund.ku.dk); Schwartz, T.W. (tws@sund.ku.dk). 0165-6147/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.08.006 Trends in Pharmacological Sciences, December 2010, Vol. 31, No. 12 567