Surface masking shapes the traffic of the neuropeptide Y Y2 receptor.
The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.
Department of Microbiology and Molecular Cell Sciences, University of Memphis, Memphis, TN 38152, USA.
Michael S
MS
Parker
Renu
R
Sah
Steven L
SL
Parker
D000595:Amino Acid Sequence; D000818:Animals; D001152:Arsenicals / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells; D002448:Cell Adhesion; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D004072:Digitonin / Q000378:metabolism / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005372:Filipin / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006168:Guinea Pigs; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D019894:Peptide YY / Q000494:pharmacology / Q000502:physiology; D037342:Pertussis Toxin / Q000494:pharmacology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D017476:Receptors, Neuropeptide Y / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D013501:Surface-Active Agents / Q000494:pharmacology
D001152:Arsenicals; D002614:Chelating Agents; D017476:Receptors, Neuropeptide Y; D013501:Surface-Active Agents; C095889:neuropeptide Y2 receptor; C029341:oxophenylarsine; D019894:Peptide YY; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005372:Filipin; D004492:Edetic Acid; D037342:Pertussis Toxin; D004072:Digitonin