The polypeptide chain of most GPCRs is post-translationally modified including N-glycosylation, palmitoylation, and phosphorylation. Potential N-glycosylation sites (NXS/T) and O-glycosylation sites (Sadeghi and Birnbaumer 1999; Nakagawa etal. 2001) are usually located within the extracellular N-terminal region, but are also found in the extracellular loops. The number and exact positions of glycosylation sites are usually not conserved among orthologs of different species. Some GPCRs, for example, the A2 adenosine and the human a2B adrenergic receptors, completely lack consensus sites for glycosylation in their N termini but are fully active in the absence of this post-translational modification. The functional relevance of post-translational modifications in GPCRs has been extensively studied in in vitro systems. It is well accepted that mutational disruption of potential N-glycosylation of most GPCRs has little effect on receptor function in vitro (Rands etal. 1990; Sadeghi and Birnbaumer 1999; Zeng et al. 1999). However, non-glycosylated receptors for parathyroid hormone and glycoprotein hormones in which glycosylation sites were mutated, are deficient in function (Zhang et al. 1995; Zhou et al. 2000). In the human calcium-sensing receptor, eight out of 11 potential N-linked glycosylation sites are actually utilized. Glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction (Ray et al. 1998). Interestingly, substitution mutations altering potential glycosylation sites in rhodopsin were described in families with retinitis pigmentosa (Bunge et al. 1993; Sullivan et al. 1993). These findings underline the functional importance of GPCR glycosylation in vivo.
Consensus acceptor phosphorylation sites for protein kinases A and C and potential receptor-specific kinase phosphorylation sites (multiple serine and threonine residues) are present in the i3 loop and the C-terminal domain. Studies with the ß2-adrenergic receptor indicated that the selectivity of receptor/G protein coupling can be regulated by receptor phosphorylation (Daaka et al. 1997). Most GPCRs also contain one or more conserved cysteine residues within their C-terminal tails (Fig. 1.3) which are modified by covalent attachment of palmitoyl or isoprenyl residues (Bouvier et al. 1995; Hayes et al. 1999). Depending on the specific GPCR examined, different effects on receptor phosphorylation, internalization, trafficking, and G protein-coupling profile have been described (reviewed in Bouvier etal. 1995; Wess 1998). However, several Family 1 GPCRs do not have Cys residues in their C-terminal tails for post-translational modification.
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