Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.

Abstract

Muscarinic acetylcholine receptors from the avian central nervous system were examined for developmental changes that correlated with the differentiation of cholinergic synapses. In contrast to previous studies that showed a single molecular weight form of muscarinic receptors in the mature central nervous system, the current study of receptors from embryonic and newly hatched chick retina showed the presence of two electrophoretic forms having apparent molecular weights of 86,200 ± 400 and 72,200 ± 300. Two receptor forms also were observed in embryonic cerebrum, optic tectum, and cerebellum. Each form was present, although decreased in molecular weight by 6000, after treatment with deglycosylating enzymes, consistent with molecular differences occurring in the protein portions, rather than the carbohydrate portions, of the molecules. The relative proportions of the high and low molecular weight receptors in retina showed a striking inversion during development. Before synaptogenesis, receptors were mainly of Mr 86,000, whereas after synaptogenesis, receptors were mainly of Mr 72,000. Development of a predominantly low molecular weight receptor population also occurred in aggregate, but not monolayer, cell culture, suggesting a possible role for cell-cell interactions in triggering the change. Pulse-chase labeling of receptors on cultured cells indicated that both forms were present on the cell surface; the labeled Mr 86,000 population had a half-life of 5 hr, whereas the labeled Mr 72,000 population had a half-life of 19 hr. The change in size of muscarinic receptors during development may reflect the action of regulatory mechanisms critical to the proper assembly and function of synapses in the central nervous system. An important goal in neurobiology is to characterize the molecular events associated with synaptic development and plasticity. Considerable progress towards this goal has come from studies of nicotinic acetylcholine receptors during formation of the neuromuscular junction. Soon (11), whereas in chicken heart, newly synthesized receptors only gradually become coupled to inhibition of adenylate cyclase (12). Recent studies of the developing avian retina have shown that the muscarinic receptor system, although functional well before synapses appear, continues to undergo biochemical differentiation during the period when synapses form. Agonist binding affinity increases and the isoelectric point of the receptor increases from 4.25 to 4.50, consistent with the possibility that molecular alteration may be involved in maturation of the system (13).Results presented here extend these studies of muscarinic receptors in the developing central nervous system. They show that muscarinic receptors have two molecular forms, Mr 86,000 and Mr 72,000, and that the predominant form changes with development. The possibilities that the high molecular weight form represents a proreceptor or, alternatively, that the two receptors are products of different genes or differentially processed RNA are discussed....

Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick

“…The apparent molecular masses of the chick mAChRs closely match those revealed in retina by previous affinity alkylation and immunoblotting analyses (Large et al, 1985;McKinnon and Nathanson, 1995). We detected immunoreactive cm2 in tissue extracts of RPE, choroid, and ciliary body despite the fact that we were unable to detect it in tissue sections.…”

“…Large et al (1985) reported that the lower molecular mass mAChR isoform (likely to be cm2) was less abundant than the larger isoforms (likely to be the sum of cm3 and cm4) in the retina of embryonic chicks, whereas the smaller mAChR isoform was more abundant than the larger isoform in hatched chicks. In contrast, McKinnon and Nathanson (1995) reported that cm4 mRNA and protein were more abundant than those of other isoforms in both embryonic and hatched chick retina and that expression levels decreased as development progressed.…”

“…Four avian mAChR subtypes have been characterized and named according to sequence homology with their mammalian counterparts: cm2 (Tietje and Nathanson, 1991), cm3 (Gadbut and Galper, 1994), cm4 (Tietje et al, 1990), and cm5 (Creason et al, 1995). Autoradiographic studies have revealed muscarinic binding sites in the IPL (Sugiyama et al, 1977;Large et al, 1985), but the neurons that express these receptors remain unknown. In addition, cm2, cm3, and cm4 receptor mRNAs and proteins have been detected by using solution hybridization, immunoblotting, and immunoprecipitation techniques in retinal tissue extracts from embryonic and hatched chicks (McKinnon and Nathanson, 1995), but the precise anatomical distribution of each isoform remains unknown.…”

Glutamate receptors modulate sodium‐dependent and calcium‐independent vitamin C bidirectional transport in cultured avian retinal cells

“…The chick retina is an useful model for neurochemical and developmental studies in the CNS since it is accessible during development and several of its neurotransmitter systems have already been well characterized (de Mello 1978; Large et al. 1985; do Nascimento et al.…”

Expression of a conserved cell-type-specific protein in nerve terminals coincides with synaptogenesis.

“…Upon contact with their target cells, growing axons induce specific events that contribute to the maturation of the postsynaptic compartment of the synapse (29)(30)(31). Similarly, phenotypic differentiation can be determined by interactions between neurons and their targets (21,32); however, a specific role for the target cell in the functional differentiation of the nerve terminal has yet to be demonstrated.…”