INFLUENCE OF 2ND AND 3RD CYTOPLASMIC LOOPS ON BINDING, INTERNALIZATION, AND COUPLING OF CHIMERIC BOMBESIN M3 MUSCARINIC RECEPTORS/

In order to investigate the molecular basis for differences in the cha racteristics of bombesin (Bn) and m3 muscarinic cholinergic (m3 ACh) r eceptors, chimeric Bn receptors possessing cytoplasmic domains from th e m3 ACh receptor were produced. The receptors were expressed in CHO-K I cells and binding, structural, and signal transduction characteristi cs 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 I-125-bombesin with a single affinity that was approximately th e same as that of the Bn receptor (5-10 nM). However, Bn receptors pos sessing the m3 ACh third cytoplasmic loop were severely affected in ot her respects, Internalization of ligand in Bn and BM2L cells was rapid and extensive (>80% of bound I-125-bombesin was acid-resistant). In c ontrast, internalization was dramatically reduced in BM3L and BM23L ce lls (similar to 20% of bound I-125-bombesin was acid-resistant). In Bn or BM2L cells 10 nM bombesin stimulated similar to 10-fold increases in phosphatidylinositol hydrolysis. Activation of Bn receptors also in duced an increase in arachidonic acid release (478 +/- 32% of control, n = 3) and large increases in intracellular Ca2+. In contrast, in BM3 L or BM23L cells, bombesin had no significant effect on phosphatidylin ositol hydrolysis. Furthermore, BM3L receptor activation did not incre ase arachidonic acid release. However, BM3L and BM23L cells showed a s mall increase in intracellular Ca2+ at high concentrations of bombesin . These data indicate that the third cytoplasmic loop alone, or togeth er with the second cytoplasmic loop, was not sufficient to transfer th e characteristics of G protein interaction between m3 ACh and bombesin receptors. Furthermore, for the Bn receptor, ligand internalization d oes, whereas formation of the high affinity binding state does not, ap pear to require activation of G proteins.