THE ROLE OF MEMBRANE PROXIMAL THREONINE RESIDUES CONSERVED AMONG GUANINE-NUCLEOTIDE-BINDING-PROTEIN-COUPLED RECEPTORS IN INTERNALIZATION OFTHE M4 MUSCARINIC ACETYLCHOLINE-RECEPTOR

Many guanine-nucleotide-binding-protein-coupled receptors contain cons ensus sequences for phosphorylation by cAMP-dependent protein kinase ( PKA), often located in the membrane proximal regions critically import ant for receptor signalling. In the present study. we have evaluated b y site-directed mutagenesis the role of the putative PKA phosphorylati on sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr 145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalizati on. Antagonist binding was unaltered by any of the mutations studied, while the agonist-binding affinity was either not affected (Thr145 ala nine), increased (Thr399 alanine) or decreased (Thr399 serine or aspar tic acid), m4 mAChR-mediated inhibition of adenylyl cyclase was unalte red by the mutations, except for an approximately tenfold reduced agon ist potency of the Thr399 aspartic acid mutated receptor. Agonist-indu ced receptor internalization was unaltered with Thr399 serine or aspar tic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these r esidues with alanine. These mutational effects could not be reproduced by treatment of wild-type receptor-expressing cells with the PKA inhi bitor H-8. Furthermore, maximal stimulation of cellular PKA neither af fected receptor internalization nor signalling measured as receptor-me diated Ca2+ mobilization. We conclude that the membrane proximal threo nine residues of the m4 mAChR are not required for receptor signalling , but replacement by alanine residues can significantly affect recepto r internalization, independently of PKA phosphorylation. Sequence comp arisons suggest that threonine residues at corresponding positions may be relevant to internalization of other guanine-nucleotide-binding-pr otein-coupled receptors.