Analysis of binding sites and efficacy of a species-specific peptide at rat and human neurotensin receptors

We have developed;a neurotensin analog, L-[3,1-naphthylalanine(11)]NT(8-13) . NT34, that can distinguish between rat and human neurotensin receptors, a nd exhibits more than a 100-fold difference in binding affinities and a 60- fold difference in functional coupling to phosphatidylinositol turnover. Us ing cells transfected with different numbers of the appropriate receptors, we measured the changes in phosphatidylinositol production, and then evalua ted the efficiency of receptor effector coupling based on Furchgott's desig n. The binding of NT34 at both rat and human neurotensin receptors stably e xpressed in CHO-K1 cells was to two sites, while the binding of NT was to o ne site. at the rat receptor the equilibrium dissociation constant (K-d) fo r NT34 at the high-affinity site was 0.058 nM, while that at the low-affini ty site was 3.1 nM. For the human receptor at the high-affinity site, the K -d for NT34 was 18 nM. while that at the low-affinity site was 180 nM. For both species the percentage of receptors representing the high-affinity sit e was approximate to 60-70% with 30-40% at the low-affinity site. We derive d agonist dissociation constants (K-a) for NT and NT34, which suggest that for NT34, the low-affinity site is functionally coupled to phosphatidy[inos itol turnover. Finally, we compared the relative efficacies of both compoun ds and found that NT34 was was about 2-fold and 4-fold more efficacious tha n NT in stimulating phosphatidy[inositol turnover in rat and human MT recep tors, respectively.