N. Hukovic et al., Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail, J BIOL CHEM, 274(35), 1999, pp. 24550-24558
We have previously reported that the human somatostatin receptor type 1 (hS
STR1) stably expressed in Chinese hamster ovary-Iii cells does not internal
ize but instead up-regulates at the membrane during continued agonist treat
ment (1 mu M somatostatin (SST)-14 x 22 h), Here we have investigated the m
olecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a
time-, temperature-, and dose-dependent manner to saturable levels, in int
act cells but not in membrane preparations. Although hSSTR1 was acutely des
ensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continue
d agonist exposure (22 h) restored functional effector coupling. Up-regulat
ion was unaffected by cycloheximide but blocked by okadaic acid. Confocal f
luorescence immunocytochemistry of intact and permeabilized cells showed pr
ogressive, time-dependent increase in surface hSSTR1 labeling, associated w
ith depletion of intracellular SSTR1 immunofluorescent vesicles. To investi
gate the structural domains of hSSTR1 responsible for up-regulation, we con
structed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 recepto
rs, Human SSTR5 was chosen because it internalizes readily, displays potent
C-tail internalization signals, and does not up-regulate. Like wild type h
SSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ab
ility to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 ind
uced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-
tail with that of hSSTR1 converted the chimeric receptor to one resembling
wild type hSSTR1 (poor internalization, 71% up-regulation). These results s
how that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dep
endent active process of receptor recruitment from a pre-existing cytoplasm
ic pool to the plasma membrane. It does not require new protein synthesis o
r signal transduction, is sensitive to dephosphorylation events, and critic
ally dependent on molecular signals in the receptor C-tail.