The GalRI receptor

In both rats and humans, GalR1 is composed of 349 amino acids, in mouse 348, and in some species and cell lines even fewer (Branchek et al. 1998; Jacoby et al. 1997). The human and rat receptors share the same consensus sites for N-linked glycosylation and intracellular phosphorylation, with the exception of two additional phosphorylation sites in the human receptor.

Table 16.3 Galanin receptors

GalR1

GalR2

GalR3

Alternative names

GN-1

Preferred endogenous ligand

Galanin

GALP

Structural information (Accession no.)

h 3 49 a a ( P4 2 71 1 ) r 346 aa (Q62805) m 348aa (P56479)

h 387 aa (O43 603) r 372 aa (O88854) m 371 aa (008726)

h 3 68 a a (0607 55) r 370aa (O88626) m 370aa (O88853)

Chromosomal location

18 q 23

1 7q2 5 .3

22 q 13. 1

Selective agonists

Galanin 1-29

(partially selective) galanin 2-29, [D-Trp]-galanin 1-29, galanin 2-11

(Partially selective) galanin 2-29

Selective antagonists

M15= M35

M35 > M15

Radioligands

[125|]-galanin

[125|]-galanin

[125 |]-galanin

G protein coupling

G,/Go

G !/Go/Gq/G1 1

G,/Go

Expression profile

Hypothalamus, amygdala, hippocampus, thalamus, brainstem, spinal cord, DRG

Cerebral cortex, hypothalamus, hippocampus, dentate gyrus, amygdala, mammiliary nuclei, cerebellum, DRG, pituitary, vas deferens, prostate, uterus, ovary, stomach, intestine, pancreas

Cerebral cortex, hypothalamus, medulla oblongata, caudate putamen, cerebellum, spinal cord, pituitary, heart, spleen,testis, liver, kidney, stomach, adrenal cortex, lung, uterus, vas deferens, choroid plexus

Physiological function

Regulation of horomone and neurotransmitter release

Regulation of the release of prolactin and growth hormone

Unclear

Knockout phenotype

Altered parasympathetic tone, enhanced hippocampal excitability, seizures

Disease relevance

Neurodegeneration, epilepsy, stroke, eating disorders, cognitive decline, pain, depression, gut motility, diabetes

Neurodegeneration, epilepsy, stroke, eating disorders, cognitive decline, pain, depression, diabetes

Eating disorders, pain, depression, diabetes

In humans, GalR1 has been mapped to chromosome 18q23, and in mouse to 18E4, which is homologous with the human chromosomal position (Jacoby et al. 1997; Nicholl et al. 1995). The human, as well as the mouse, GalR1 receptor gene contains three exons, where exon 1 encodes the NH2-terminal end and the first five transmembrane (TM) domains, exon 2 the third extracellular loop, and exon 3 the remainder of the receptor, from TM 6 to the COOH-terminus (Iismaa et al. 1998).

The interactions between galanin and the GalR1 receptor were studied by mutagenesis studies of the GalR1 receptor, and by use of galanin analogues in which the side chains of the amino acids that were assumed to interact with the receptor were substituted (Fig. 16.3) (Berthold etal. 1997; Kask etal. 1996). Using computer modelling and the rhodopsin scaffold for the GalR1 and a mutagenesis approach, galanin was docked into the GalR1 receptor, with interactions between Trp2 of the ligand and His264 or His267 of the receptor, with Phe282 of the receptor and Tyr9 in galanin, and through a hydrogen bond between Glu271 of the receptor and the N-terminus of galanin (Fig. 16.4). Subsequent mutagenesis studies indicated that the N-terminus of galanin might also interact with Phe115 in TMIII (Berthold et al. 1997). The assumed interactions accounted for about 80 per cent of the observed binding free energy of the galanin-GalR1 interactions, although the model suggested that the C-terminal half of the galanin molecule is not in contact with the receptor. This again suggests that in the case of the GalR1 subtype it is the N-terminal portion of galanin that is important for binding to the receptor, and the extracellular domains of the receptor are most important for binding of the peptide ligand, as expected. It is noteworthy that in this model the N-terminus

Fig. 16.3 A large number of residues in the human GalR1 were mutated into alanines, or in some cases other amino acids. This initial mutagenesis study showed that His264, His267, and Glu271 at the top of TMVI, and Phe282 in TMVII were important for binding of galanin to the receptor.

Fig. 16.4 Computer model based on the coordinates for rhodopsin, with the galanin molecule docked into its binding site at the human GalRl. In the proposed model, the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+-sensitive pair of His264 and His267 of TMVI, and Tyr9 of galanin interacts with Phe282 of TMVII, while the C-terminus of galanin is pointing towards the N-terminus of the receptor. Subsequent mutations of residues in the receptor indicated that Phe115 in TMIII might also interact with the N-terminus of galanin.

Fig. 16.4 Computer model based on the coordinates for rhodopsin, with the galanin molecule docked into its binding site at the human GalRl. In the proposed model, the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+-sensitive pair of His264 and His267 of TMVI, and Tyr9 of galanin interacts with Phe282 of TMVII, while the C-terminus of galanin is pointing towards the N-terminus of the receptor. Subsequent mutations of residues in the receptor indicated that Phe115 in TMIII might also interact with the N-terminus of galanin.

of the receptor does not interact with galanin, and the interactions are concentrated to the top of transmembrane domains III, VI, and VII (Figs 16.3 and 16.4). Glycosylation of the receptor does not seem to be important for binding of galanin.

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