Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells.
The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.
Jans
D A
DA
Max-Planck-Institut für Biophysik, Frankfurt am Main, Federal Republic of Germany.
Peters
R
R
Jans
P
P
Fahrenholz
F
F
D000199:Actins / Q000032:analysis; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D011945:Receptors, Angiotensin / Q000378:metabolism*; D017483:Receptors, Vasopressin; D015398:Signal Transduction; D013696:Temperature; D014667:Vasopressins / Q000378:metabolism*
D000199:Actins; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D014667:Vasopressins; D001127:Arginine Vasopressin; D005576:Colforsin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases