Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies

Abstract

Mouse monoclonal antibodies were raised against bacterially expressed protein sequences of the NR2A, NR2B, NR2C and NR2D subunits of the rat NMDA receptor. From immunoblots of rat brain proteins, the apparent molecular weights of these subunits were 165, 170, 135 and 145 kDa, respectively. Proteins of similar masses were observed on immunoblots of specifically transfected HEK293 cells. Deglycosylation with endoglycosidase F reduced the mass of each endogenous NR2 subunit by ≈10 kDa. In distribution studies, NR2A-immunoreactive protein (IRP) was located throughout the adult rat brain, NR2B-IRP was primarily in the forebrain, NR2C-IRP was predominantly in the cerebellum and NR2D-IRP was mainly found in the thalamus, midbrain and brainstem. Whereas NR2A- and NR2C-IRPs increased during rat brain post-natal development, NR2B- and NR2D-IRPs were abundant at birth and declined with age, especially in cerebellum. NR2-IRPs of mouse, rabbit, frog and human brain were of sizes similar to those of the corresponding rat subunits and were similarly distributed. In summary, NR2 subunits are large glycoproteins whose specific expression profiles in the brain are developmentally and regionally regulated and which are similarly expressed in a variety of species.

Introduction

The N-methyl-d-aspartate (NMDA) type of ionotropic glutamate receptor is physiologically important in excitatory neurotransmission and synaptic plasticity, and pathologically important in excitatory ischaemic neural death (see [33]for review). The rat, mouse and human genes encoding subunits of this receptor have been identified and classified into two related groups: the NMDAR1 (NR1 or ζ) subunit 20, 36, 41, 56and the NMDAR2 subunits A, B, C and D (NR2A–D or ϵ1–4) 17, 18, 23, 29, 34. Small channel responses can be detected from homomeric NR1 receptors recombinantly expressed in oocytes [36]but co-expression of NR2 subunits greatly enhances responses 18, 34. Thus, endogenous NMDA receptors are believed to be heteromeric assemblies containing NR1 subunits co-assembled with one, or perhaps more, type(s) of NR2 subunit 4, 9, 46, 52. The NR1 subunit is essential for NMDA receptor function since its absence prevents classical NMDA receptor responses both in recombinant expression systems [34]and in neurones cultured from the brains of mice lacking the NR1 gene [13].

Some heterogeneity of receptor function may be achieved by alternative splicing of the NR1 subunit mRNA. The insertion of a highly charged 21 amino-acid cassette into the N-terminal region of homomeric NR1 channels expressed in Xenopus oocytes prevents current potentiation by Zn²⁺ ions, alters pH sensitivity and mildly affects agonist and antagonist sensitivities while alternative C-termini produced by sequential cassette deletions may affect the regulation of NMDA channel function by protein kinase C 11, 12, 15, 49, 50, 56. The influences of such alternative splicing on the function of recombinant heteromeric NMDA receptors has yet to been studied in detail. However, the NR2 subunit of recombinant heteromeric NR1–NR2 NMDA receptors exerts a large influence on receptor functions, such as channel kinetics, sensitivity to Mg²⁺ blockade and ligand affinities 6, 18, 23, 26, 34, 35. The results of such studies on heteromeric recombinant NMDA receptors, considered together with the CNS distributions of the mRNAs encoding the NR2 subunits 18, 34, 35and NR1 splice variants 27, 28, 47, strongly suggest that the regional and developmental heterogeneity of endogenous NMDA receptor properties [33]is a consequence of regionally and developmentally different NR1–NR2 subunit combinations.

However, the confidence in NR1 and NR2 mRNA expression as a measure of subunit abundance has recently been undermined by the observation that cultured phaeochromocytoma PC12 cells and neuroblastoma Y79 cells express NMDA receptor subunit mRNAs but not the encoded proteins 21, 48. The distribution and properties of the endogenous subunit proteins should therefore be confirmed directly. Rabbit polyclonal antibodies have recently been raised against the rat NR1 splice variants 2, 5, 8, 39, 46and the four NR2 subunits 4, 21, 42, 46, 53, 55and have been used to examine properties and expression of the NR1 and NR2 antigens. In these reports, generally one or two NR2 subunits were compared and only one study [54]simultaneously compared the regional adult expression of all four NR2 subunits. A comprehensive, simultaneous examination of all four rat NR2 subunits, their regional and developmental expression and their properties in other species has not yet been published. Mouse monoclonal antibodies against the NR2 subunits have also not yet been reported. As part of our continuing investigations on glutamate-gated ion channels, we have generated mouse monoclonal antibodies specific for the N- and C-terminal regions of the four rat NR2 subunits and used them to study properties of these proteins. Part of this study has been presented in abstract form [25].