Stoichiometric analysis of internalization, recycling, and redistribution of photoaffinity-labeled guanylate cyclase/atrial natriuretic factor receptors in cultured murine Leydig tumor cells.
The membrane-bound form of guanylate cyclase represents a biologically active atrial natriuretic factor receptor (GC/ANF-R). Murine Leydig tumor (MA-10) cells predominantly overexpress GC/ANF-R in high density (Pandey, K. N., Pavlou, S. N., and Inagami, T. (1988) J. Biol. Chem. 263, 13406-13413; Pandey, K. N., and Singh, S. (1990) J. Biol. Chem. 265, 12342-12348). Information regarding the post-binding events of GC/ANF-R is obscure. This study presents the kinetics of internalization, recycling, and redistribution of GC/ANF-R in model MA-10 cells. Both the 125I-ANF binding assays and photoaffinity labeling procedures were utilized to label the total, intracellular, and cell surface GC/ANF-R. After the binding of 125I-ANF to GC/ANF-R, this complex was internalized and both the intact and degraded ligands were released into culture media. The distribution of 125I-ANF on the cell surface, in the intracellular compartments, and into culture media provided a dynamic relationship between the rates of 125I-ANF uptake, its degradation, and extrusion. The extent of receptor recycling was measured using tryptic proteolysis of photoaffinity-labeled GC/ANF-R to distinguish cell surface receptors from those that were internalized. A population of GC/ANF-R rapidly recycled (t1/2 = 5 min) from intracellular compartment to plasma membrane. Recycling of GC/ANF-R was impaired by chloroquine, dinitrophenol, and low temperature (22 degrees C). Furthermore, these studies suggest that dissociation of ANF from the receptor is not required for recycling of internalized GC/ANF-R.
Pandey
K N
KN
Department of Biochemistry and Molecular Biology, Medical College of Georgia, School of Medicine, Augusta 30912.
D000345:Affinity Labels; D000818:Animals; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D007984:Leydig Cell Tumor; D046911:Macromolecular Substances; D051379:Mice; D010777:Photochemistry; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D046911:Macromolecular Substances; D002738:Chloroquine; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor