Essential role of dynamin in internalization of M-2 muscarinic acetylcholine and angiotensin AT(1A) receptors

Most G protein-coupled receptors (GPCRs), including the M-1 muscarinic acet ylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a proc ess that requires dynamin GTPase. The observation that some GPCRs like the M-2 mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespe ctive of expression of dominant-negative K44A dynamin has led to the propos al that internalization of these GPCRs is dynamin-independent. Here, we rep ort that, contrary to what is postulated, internalization of M-2 mAChR and AT(1A) HEK-293 cells is dynamin-dependent, Expression of N272 dynamin, whic h lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4,5-bisphosphate, strongly inhibits internalizatio n of M-1 and M-2 mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) redu ces M-1 mAChR internalization. Similarly, c-Src inhibitor PP1 as well as th e generic tyrosine kinase inhibitor genistein strongly inhibit M-1 aAChR in ternalization. In contrast, M-2 mAChR internalization is not (or is only sl ightly) reduced by expression of these constructs or treatment with PP1 or genistein, Thus, dynamin GTPases are not only essential for M-1 mAChR but a lso for M-2 mAChR and AT(1A)R internalization in HEK-293 cells. Our finding s also indicate that dynamin GTPases are differentially regulated by c-Src- mediated tyrosine phosphorylation.