Role of the domain encompassing Arg304-Ile328 in rat P2X2 receptor conformation revealed by alterations in complex glycosylation at Asn298.
The final 25 amino acids of the ectodomain of the P2X receptors, immediately prior to the second TM (transmembrane domain) (pre-TM2: Arg(304)-Ile(328) in rat P2X(2)), are highly conserved. Whole-cell patch clamp recordings showed that single cysteine substitutions in the N-terminal half of pre-TM2 (Arg(304)-Ile(314)) led to loss of function at Arg(304), Leu(306), Lys(308) and Ile(312). Cysteine substitutions within this region also resulted in a significant reduction in the apparent molecular mass of receptors, due to loss of complex glycosylation at the nearby acceptor site Asn(298), which was not seen for the C-terminal portion of pre-TM2 (Asp(315)-Ile(328)). The reduction in complex glycosylation was not due to reduced cell-surface presentation, demonstrating that glycosylation at Asn(298) was acting as a sensor of subtle changes in receptor conformation within the pre-TM2 region. When this N-glycan site was repositioned closer to the plasma membrane by mutagenesis (N298S together with G299N, T300N, T301N or T303N), glycosylation was restored at G299N and T300N, but was impaired for T301N and completely absent for T303N. These results suggest that the region in the vicinity of Asp(315) is at the plasma membrane interface and that the N-terminal portion of pre-TM2 (Arg(304)-Ile(314)) is important for the correct conformation of the receptor at the extracellular face of the membrane.
Young
Mark T
MT
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, UK. mark.young@manchester.ac.uk
Zhang
Yi-Hong
YH
Cao
Lishuang
L
Broomhead
Helen
H
Jiang
Lin-Hua
LH
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000196:embryology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D058476:Receptors, Purinergic P2X2
C546401:P2RX2 protein, human; C546403:P2rx2 protein, rat; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D001216:Asparagine