Open AccessThe Role of Membrane Proximal Threonine Residues Conserved among Guanine‐Nucleotide‐Binding‐Protein‐Coupled Receptors in Internalization of the m4 Muscarinic Acetylcholine Receptor
Author(s) -
Koppen Chris J.,
Lenz Wolfgang,
Nunes J. Pedro L.,
Zhang Chunyi,
Schmidt Martina,
Jakobs Karl H.
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.536_b.x
Subject(s) - internalization , biochemistry , alanine , muscarinic acetylcholine receptor m5 , biology , receptor , chemistry , muscarinic acetylcholine receptor m3 , muscarinic acetylcholine receptor , amino acid
Many guanine‐nucleotide‐binding‐protein‐coupled receptors contain consensus sequences for phosphorylation by cAMP‐dependent protein kinase (PKA), often located in the membrane proximal regions critically important for receptor signalling. In the present study, we have evaluated by site‐directed mutagenesis the role of the putative PKA phosphorylation sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalization. Antagonist binding was unaltered by any of the mutations studied, while the agonist‐binding affinity was either not affected (Thr145 alanine), increased (Thr399 alanine) or decreased (Thr399 serine or aspartic acid). m4 mAChR‐mediated inhibition of adenylyl cyclase was unaltered by the mutations, except for an approximately tenfold reduced agonist potency of the Thr399 aspartic acid mutated receptor. Agonist‐induced receptor internalization was unaltered with Thr399 serine or aspartic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these residues with alanine. These mutational effects could not be reproduced by treatment of wild‐type receptor‐expressing cells with the PKA inhibitor H‐8. Furthermore, maximal stimulation of cellular PKA neither affected receptor internalization nor signalling measured as receptor‐mediated Ca 2+ mobilization. We conclude that the membrane proximal threonine residues of the m4 mAChR are not required for receptor signalling, but replacement by alanine residues can significantly affect receptor internalization, independently of PKA phosphorylation. Sequence comparisons suggest that threonine residues at corresponding positions may be relevant to internalization of other guanine‐nucleotide‐binding‐protein‐coupled receptors.