Molecular characterization

In 1987, Servin et al. managed to identify and biochemically characterize a galanin-galanin receptor binding complex in the rat brain, which on SDS-PAGE was revealed as a 56 kD protein (Amiranoff et al. 1989; Fisone et al. 1989; Servin et al. 1987), suggesting that it may be a GPCR, as it indeed also turned out to be, as shown in cloning studies later (see below).

So far, three subtypes of the galanin receptor, a typical 7-transmembrane domain G protein-coupled receptor, have been cloned and named GalR1, GalR2, and GalR3, respectively (Table 16.3) (Branchek et al. 2000). The three receptor subtypes differ in terms of their primary sequence, genomic organization, pharmacological profile, functional coupling, and

Table 16.2 Displacement binding of galanin and galanin fragments in the rat CNS

1 16

29 IC50 (nM)

(1-29)

GWTLNSAGYLLGPHAIDNHRSFHDKYGLA 0.8

(1 -1 6)

7.0

(17-29)

----------------- >10000

(1 -1 3 )

150

[AC-GLY1

] -( 1-1 6)

500

[Ala1 ](1-'

6)

A-----------------

90

[Ala2 ](1-'

6)

-A----------------

>10000

[Ala3 ](1-'

6)

- -.A --------------

230

[Ala4](1-'

6)

- - -.A-------------

90

[Ala5](1-

6)

- - - -A ------------

1900

[Ala6](1-'

6)

----- A -----------

60

[Ala8](1-'

6)

-------- A --------

200

[Ala9](1-'

6)

--------- A -------

1900

[Ala10](1-

-1 6)

---------- A ------

2400

[Ala11 ](1-

-1 6)

-----------A-----

1200

[Ala12 ](1-

-1 6)

------------ A - ---

1000

[Ala13 ](1-

-1 6)

------------- A ---

80

[Ala14](1-

-1 6)

--------------A--

20

[Ala16](1-

-1 6)

---------------- A

50

distribution . These variations all contribute to the ligand selectivity, signalling divergence, and specific importance of each respective subtype in various systems .

The first galanin receptor (GalRl) was cloned by Habert-Ortoli et al. from Bowes human melanoma cell line (Habert-Ortoli et al. 1994), a little more than a decade after the discovery of the endogenous ligand galanin This discovery was followed by the cloning of the rat GalRl homologue in 1995, and two more subtypes (GalR2 and GalR3) have since then also been cloned from several species (Ahmad etal. 1998; Bloomquist etal. 1998; Borowsky etal. 1998; Burgevin et al. 1995; Fathi et al. 1997, 1998a; Howard et al. 1997; Iismaa et al. 1998; Jacoby etal. 1997; Kolakowski etal. 1998; Lorimer andBenya 1996; Lorimer etal. 1997; Pang etal. 1998; Parker etal. 1995; Smith etal. 1997; Sullivan etal. 1997; Wang etal. 1997a-c).

For each galanin receptor subtype there is a high sequence homology between different species Between receptor subtypes however, the sequence similarity is lower; the three receptors reveal only around 35-50 per cent amino acid identity. In addition, the GalR1 gene

Pro 13

Pro 13

Fig. 16.2 Distribution of galanin-positive cell groups and galanin-binding sites in the rat brain.

has a different intron/exon organization, as compared to the genes for GalR2 and GalR3 (Iismaa et al. 1998; Kolakowski et al. 1998), pointing at, at least two different paths for galanin receptors throughout evolution. The highest homology between the three different galanin receptor subtypes is found in the transmembrane domains, while the extracellular and intracellular regions display a lower degree of similarity. This is in good agreement with the suggestion that the latter are responsible for selective binding of ligands, and for differential G protein coupling and signalling pathways (Berthold and Bartfai 1997).

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