Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors

Galanin receptors (GALRs) belong to the superfamily of G-protein coupled receptors. The three GALR subtypes (GALR1, GALR2, and GALR3) are activated by their endogenous ligands: spexin (SPX) and galanin (GAL). The synthetic SPX-based GALR2-specific agonist, SG2A, plays a dual role in the regulation of appetite and depression-like behaviors. Little is known, however, about the molecular interaction between GALR2 and SG2A. Using site-directed mutagenesis and domain swapping between GALR2 and GALR3, we identified residues in GALR2 that promote interaction with SG2A and residues in GALR3 that inhibit interaction with SG2A. In particular, Phe¹⁰³, Phe¹⁰⁶, and His¹¹⁰ in the transmembrane helix 3 (TM3) domain; Val¹⁹³, Phe¹⁹⁴, and Ser¹⁹⁵ in the TM5 domain; and Leu²⁷³ in the extracellular loop 3 (ECL3) domain of GALR2 provide favorable interactions with the Asn⁵, Ala⁷, Phe¹¹, and Pro¹³ residues of SG2A. Our results explain how SG2A achieves selective interaction with GALR2 and inhibits interaction with GALR3. The results described here can be used broadly for in silico virtual screening of small molecules for the development of GALR subtype-specific agonists and/or antagonists.