Mutagenesis and Ligand Modification Studies on Galanin Binding to its GTP‐Binding‐Protein‐Coupled Receptor GalR1

Abstract

In this study, a large number of receptor mutants were generated and several N-terminally modified galanin analogues synthesized to refine the previously proposed binding site model for galanin to its GTP-binding-protein-coupled receptor GalRl . In addition to ligand-binding studies, the functionality of mutant receptors was evaluated by assessing their ability to mediate galaninergic inhibition of isoproterenol-stimulated adenylyl cyclase activity. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remain functional albeit 20-fold less efficient than the wild-type receptor in mediating inhibition of stimulated CAMP production by galanin. The His267Ala mutant is, apart from being deficient in galanin binding, also severely impaired in functional coupling. While His264 and Phe282 seem to be important in forming the binding pocket for galanin, His267 might play a role in forming or stabilizing the active conformation of the GalRl receptor rather than directly participating in the formation of the binding pocket for galanin.N-terminal carboxylic acid analogues of galanin have low affinity to wild-type GalRl, but rubstantially increased affinity to the Glu27lLys receptor mutant. This, together with the finding that an alanine substitution of PhellS in TM 111 results in a tenfold decrease in affinity for galanin, suggests that the N-terminus of galanin interacts with PhellS. In contrast to the Phe282AIa mutation in TM VII, a conservative mutation of Phe282 to tyrosine did not alter the affinity for galanin. Thus, the interaction between Tyr9 of galanin and Phe282 is likely to be of an aromatic-aromatic nature.Keywords: galanin ; mutagenesis ; receptor ; binding ; peptide.The neuropeptide galanin plays important roles in physiological functions such as pain signalling, acquisition, feeding, and hormone release (for reviews, see [2, 31). The effects are mediated via 7-transmembrane (7-TM) spanning domain type receptors coupled via inhibitory GTP-binding proteins (GJG,) Structure/activity relationship studies using galanin fragments and analogues show that high-affinity binding as well as agonist action resides in the N-terminal part of galanin, which in its first 14 amino acids is fully conserved in all species studied. Studies of L-Ala substituted analogues of galanin demonstrate that Trp2 and Tyr9 as well as the free N-terminal amino group are the major pharmacophores [S].The ligand-binding sites of GTP-binding-protein-coupled receptors for small ligands such as monoamines [9] and low-molecular-mass, non-peptide antagonists to peptide receptors [ 101 have been delineated in recent years. These small ligands, with molecular masses of less than 500 Da, bind at sites in the TM domains (monoamines) or near the extracellular tops of these helices (non-peptide antagonists) [lo]. Recently, studies on the binding mode of intermediately sized peptides such as substance Neurotoxicology, Stockholm University, S-I 06 91 Stockholm Sweden P and angiotensin I1 h...

Determining receptor–ligand interaction of human galanin receptor type 3

“…Previous studies of hGalR1 have shown that a Phe115 3.32 Ala exchange affect the interaction between the receptor and galanin and Phe115 3.32 was suggested to interact with Gly1 in galanin (Berthold et al, 1997). In contrast, the Phe106 3.33 Ala mutation in hGalR2 did not affect galanin binding (Lundströ m et al, 2007).…”

“…Identification of receptor residues that are involved in ligand binding is a fundamental step in elucidating and understanding the principles of ligand-receptor interactions. Such studies have been performed on GalR1 and GalR2, delineating both conserved and subtype different interaction with galanin receptor ligands (Kask et al, 1996;Berthold et al, 1997;Kask et al, 1998;Church et al, 2002;Lundströ m et al, 2007).…”

“…E-model is the sum of the Glide Score, ligand-receptor molecular mechanics interaction energy and the ligand strain energy (Friesner et al, 2004). Presence of ligand contacts with conserved residues found as important in GalR1 and GalR2 studies (Berthold et al, 1997;Kask et al, 1998;Church et al, 2002;Lundströ m et al, 2007) was additionally accounted. Low energy clusters with members showing preference in binding to these residues were preferred.…”

“…Site-directed mutagenesis studies have been performed on both hGalR1 and hGalR2 to map the binding pocket (Kask et al, 1996;Berthold et al, 1997;Kask et al, 1998;Lundströ m et al, 2007), and a three-dimensional model of hGalR1 has been presented by Iismaa and co-workers (Church et al, 2002). Altogether these reports indicate that TM helices III, VI and VII together with extracellular loop (ECL) II and III form the binding pocket of Galanin is a neuropeptide found throughout the central and peripheral nervous systems of a wide range of species, ranging from human and mouse to frog and tuna.…”

Galanin Protects Against Behavioral and Neurochemical Correlates of Opiate Reward

“…However, it should be noted that galnon can also interact directly with some G proteins (Flören et al, 2005), thus the action of galnon may not be limited to activation of GalR1. GalR1 is negatively coupled to adenylyl cyclase and activation of this receptor subtype can therefore decrease PKA-mediated signaling as well as neuronal excitability (Berthold et al, 1997). Thus, galnonmediated activation of GalR1 could reverse the effects of morphine on ERK signaling either by decreasing the Figure 6 Summary of neurochemical changes.…”