The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor.
The dimerization properties of the ghrelin receptor (GRLN-R) and its non-signalling, naturally occurring, truncated splice variant (GHS-R1b) have been investigated in human embryonic kidney 293 cells heterologously expressing these proteins. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the formation of GRLN-R homodimers and GRLN-R/GHS-R1b heterodimers, but ghrelin-induced conformational changes were only detected in the GRLN-R homodimers. When the expression of GHS-R1b exceeded that of GRLN-R, there was a decrease in the cell surface expression of GRLN-R with a consequent decrease in constitutive activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Furthermore, there was no change in ghrelin affinity, and the efficacy of cell signalling as measured by stimulation of PI-PLC and extracellular signal-regulated kinase 1/2 was unchanged. Cellular localization studies suggest that GRLN-R is normally distributed between the plasma membrane and cytosolic fractions, but in the presence of GHS-R1b, GRLN-R is localized to the nucleus. Therefore, we propose that the decrease in GRLN-R constitutive signalling was due to translocation of GRLN-R to the nucleus due to the formation of GRLN-R/GHS-R1b heterodimers. Therefore, GHS-R1b appears to act as a dominant-negative mutant of the full-length GRLN-R.
Leung
Po-Ki
PK
Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
Chow
Kevin B S
KB
Lau
Pui-Ngan
PN
Chu
Kit-Man
KM
Chan
Chi-Bun
CB
Cheng
Christopher H K
CH
Wise
Helen
H
D017398:Alternative Splicing; D002460:Cell Line; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054440:Receptors, Ghrelin; D014162:Transfection
D020033:Protein Isoforms; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin