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

• Tipo: Artigo em Revista Científica Internacional
• Data: 1995

Autores

VANKOPPEN, CJ (Autor) Outra; LENZ, W (Autor) Outra; José Pedro Nunes (Autor) FMUP; ZHANG, CY (Autor) Outra; SCHMIDT, M (Autor) Outra; JAKOBS, KH (Autor) Outra

Revista

Título: European Journal of Biochemistry

Vol. 234

Páginas: 536-541

ISSN: 0014-2956

Editora: Blackwell

Indexação

ISI Web of Knowledge - 10 Citações

Outras Informações

ID Authenticus: P-00G-9N1

Abstract (EN): 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 Ca2+ 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.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 6