C-terminal processing of GABARAP is not required for trafficking of the angiotensin II type 1A receptor.
OBJECTIVE
GABARAP, a small (117 aa) trafficking protein, binds to the C-terminal, cytoplasmic domain of rat angiotensin type-1A receptor (AT(1)R), the predominant effector of the octapeptide angiotensin II (Ang II) (Cook et al., Circ. Res. 2008;102:1539-47). The objectives of this study were to map the interaction domains of GABARAP and AT(1)R, to determine the effect of GABARAP association on AT(1)R signaling activity, and to determine the importance of post-translational processing of GABARAP on accumulation of AT(1)R on the plasma membrane and its signaling function.


RESULTS
Deletion analysis identified two regions within GABARAP necessary for interaction with AT(1)R in yeast two-hybrid assays: 1) a domain comprised of residues 32-51 that is nearly identical to that involved in binding and intracellular trafficking of the GABA(A) receptor and 2) a domain encompassing the C-terminal 21 aa. The GABARAP interaction domain of AT(1)R was delimited to the 15 aa immediately downstream of the last membrane spanning region. Overexpression of GABARAP in rat adrenal pheochromocytoma PC-12 cells increased the cell-surface expression of AT(1)R and Ang II-dependent activation of the cAMP signaling pathway. Residues within AT(1)R necessary for these responses were identified by mutational analysis. In PC-12 cells, GABARAP was constitutively and quantitatively cleaved at the C-terminus peptide bond and this cleavage was prevented by mutation of Gly(116). Wild-type GABARAP and the G116A mutant were, however, equally effective in stimulating AT(1)R surface expression and signaling activity.


CONCLUSIONS
GABARAP and AT(1)R interact through discrete domains and this association regulates the cell-surface accumulation and, consequently, ligand-induced function of the receptor. Unlike that observed with the GABA(A) receptor, this regulation is not dependent on C-terminal processing and modification of GABARAP.
Alam
Jawed
J
Laboratory of Molecular Genetics, Ochsner Clinic Foundation, New Orleans, LA 70121, USA.
Deharo
Dawn
D
Redding
Kevin M
KM
Re
Richard N
RN
Cook
Julia L
JL
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000235:genetics / Q000378:metabolism; D000818:Animals; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology*
C502186:GABARAP protein, rat; D008869:Microtubule-Associated Proteins; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II