Agonist-independent internalization and activity of a C-terminally truncated somatostatin receptor subtype 2 (delta349).
The rat somatostatin receptor subtype 2 (SSTR2) is rapidly internalized and phosphorylated in the presence of somatostatin 14 (SST14). Several C-terminal deletion constructs of SSTR2 have been investigated for their ability to undergo agonist-dependent internalization by using biochemical ligand binding assays and confocal microscopic analysis. Whereas mutant receptors lacking either 10 (delta359), 30 (delta339), or 44 (delta325) amino acid residues at the C terminus required SST14 for internalization, a construct lacking the last 20 amino acids (delta349) was detected mostly intracellularly and independently of the presence of the agonist. When internalization was blocked by sucrose, the delta349 receptor remained at the cell surface, strongly indicating that this mutant is internalized in an agonist-independent fashion. An increased affinity for agonists as measured in membrane binding assays and a reduced level of forskolin-stimulated cyclic AMP accumulation in human embryonic kidney cells expressing delta349 are properties that are characteristic of agonist-independent receptor activity. Delta349 is not phosphorylated detectably in the absence of agonist, demonstrating that phosphorylation per se is not a prerequisite for internalization of SSTR2. This observation is in line with data obtained for the delta325 mutant, which was internalized in an agonist-dependent manner, but not phosphorylated in either the presence or absence of SST14. We conclude that truncation of the SSTR2 C terminus at position 349 leads to agonist-independent, constitutive activity and internalization.
Schwartkop
C P
CP
Institut für Zellbiochemie und klinische Neurobiologie, UKE, Universität Hamburg, Germany.
Kreienkamp
H J
HJ
Richter
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017481:Receptors, Somatostatin / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D013004:Somatostatin / Q000494:pharmacology; D014982:Xenopus laevis
D010446:Peptide Fragments; D017481:Receptors, Somatostatin; D013004:Somatostatin; C093733:somatostatin receptor 2; D000242:Cyclic AMP