Influence of second and third cytoplasmic loops on binding, internalization, and coupling of chimeric bombesin/m3 muscarinic receptors

In order to investigate the molecular basis for differences in the characteristics of bombesin (Bn) and m3 muscarinic cholinergic (m3 ACh) receptors, chimeric Bn receptors possessing cytoplasmic domains from the m3 ACh receptor were produced. The receptors were expressed in CHO-K1 cells and binding, structural, and signal transduction characteristics were analyzed. Cell lines bearing chimeric Bn receptors possessing m3 ACh receptor domains in place of either the second cytoplasmic loop (BM2L), the third cytoplasmic loop (BM3L), or both loops (BM23L) each bound ¹²⁵I-bombesin with a single affinity that was approximately the same as that of the Bn receptor (5-10 nM). However, Bn receptors possessing the m3 ACh third cytoplasmic loop were severely affected in other respects. Internalization of ligand in Bn and BM2L cells was rapid and extensive (>80% of bound ¹²⁵I-bombesin was acid- resistant). In contrast, internalization was dramatically reduced in BM3L and BM23L cells (~20% of bound ¹²⁵I-bombesin was acid-resistant). In Bn or BM2L cells 10 nm bombesin stimulated ~10-fold increases in phosphatidylinositol hydrolysis. Activation of Bn receptors also induced an increase in arachidonic acid release (478 ± 32% of control, n = 3) and large increases in intracellular Ca²⁺. In contrast, in BM3L or BM23L cells, bombesin had no significant effect on phosphatidylinositol hydrolysis. Furthermore, BM3L receptor activation did not increase arachidonic acid release. However, BM3L and BM23L cells showed a small increase in intracellular Ca²⁺ at high concentrations of bombesin. These data indicate that the third cytoplasmic loop alone, or together with the second cytoplasmic loop, was not sufficient to transfer the characteristics of G protein interaction between m3 ACh and bombesin receptors. Furthermore, for the Bn receptor, ligand internalization does, whereas formation of the high affinity binding state does not, appear to require activation of G proteins.