In this study, we show that a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (D1DR) specifically inhibited D1DR binding and function, without affecting receptor oligomerization. It has been shown that an analogous peptide from the ␤2-adrenergic receptor disrupted dimerization and adenylyl cyclase activation in the ␤2-adrenergic receptor (Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. Immunoblot analyses of the D1DR revealed the receptor existed as monomers, dimers, and higher order oligomers and that these oligomeric states were unaffected after incubation with the D1DR transmembrane 6 peptide. These findings represent the first demonstration that a peptide based on the transmembrane 6 of the D1DR may represent a novel category of noncompetitive D1DR antagonists. Dopamine receptors belong to the family of G protein-coupled receptors (GPCRs)1 and mediate the many biological actions of the neurotransmitter dopamine in brain and periphery. The distinct biological functions of dopamine are mediated by the five dopamine receptor subtypes, that are divided into the D1-like (D1 and D5) and D2-like (D2, D3, and D4) subfamilies. We have previously provided evidence for the existence of dimers and oligomers of the D1 and D2 dopamine receptors (1-4). The occurrence of receptor oligomers may be an universal phenomenon in the GPCR family, as suggested by observations for serotonin 5HT-1B (5), ␤2-adrenergic and V2 vasopressin (6), metabotropic glutamate (7), ␦ opioid (8), H2 histamine (9), and D3 dopamine (10) receptors. A survey of receptor families (other than the GPCRs) reveals that these receptors are often organized into functional units that involve dimerized or oligomerized proteins. Dimerization of receptor monomers is a critical and necessary mechanism for signal transduction in tyrosine kinase receptors (11) such as the epidermal growth factor (12) and insulin receptors (13), the transferrin receptor (14), and steroid receptors (15). However, the functional significance of GPCR oligomers remains unknown. Intriguing insights from studies using receptor chimeras have suggested that both intramolecular and intermolecular interactions occur in GPCRs (16), but the role of such interactions in signal transduction is not known.Peptide sequences derived from specific regions of large proteins have been successfully studied to elucidate the function of these regions. The use of such peptide "probes" has enabled the mapping of the functional domains of certain proteins and the identification of domains that promote interactions with other proteins. This strategy has also been used in the study of GPCRs. For instance, studies have used peptides corresponding to the third cytoplasmic loops of GPCRs to elucidate the importance of this region of the receptor for interaction with G proteins. (17-19).Recently, a peptide derived from transmembrane (TM) domain 6 of the ␤2-adrenergic receptor (␤2AR) was shown to disrupt dimers of that receptor ...

The R domain of cystic fibrosis transmembrane conductance regulator (CFTR) connects the two halves of the protein, each of which possess a transmembranespanning domain and a nucleotide binding domain. Phosphorylation of serine residues, which reside mostly within the C-terminal two-thirds of the R domain, is required for nucleotide-dependent activation of CFTR chloride channel activity. The N terminus of the R domain is also likely to be important in CFTR function, since this region is highly conserved among CFTRs of different species and exhibits sequence similarity with the "linker region" of the related protein, P-glycoprotein. To date, however, the role of this region in CFTR channel function remains unknown. In this paper, we report the effects of five disease-causing mutations within the N terminus of the CFTR-R domain. All five mutants exhibit defective protein processing in mammalian HEK-293 cells, suggesting that they are mislocalized and fail to reach the cell surface. However, in the Xenopus oocyte, three mutants reached the plasma membrane. One of these mutants, L619S, exhibits no detectable function, whereas the other two, D614G and I618T, exhibit partial activity as chloride channels. Single channel analysis of these latter two mutants revealed that they possess defective rates of channel opening, consistent with the hypothesis that the N terminus of the R domain participates in ATP-dependent channel gating. These findings support recent structural models that include this region within extended boundaries of the first nucleotide binding domain.