Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human ␤2-adrenergic receptor (␤ 2AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between ␤2AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between ␤2AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the ␤2AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the ␤2AR and the regulatory protein ␤-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions. G protein-coupled receptors (GPCR) represent the single largest family of transmembrane receptors involved in cell signaling. Until recently, they were believed, unlike most other membrane receptors, to function as monomeric entities that interact with G proteins once stabilized in their active conformation by agonist binding. However, a growing body of functional and biochemical evidence suggests that they may exist as homo-or heterodimers. The functional evidence is based largely on positive and negative effects that dominant receptor mutants have on wild-type receptor function and on the observation that coexpression of two defective receptors can restore activity (1-6). More recently, coexpression of the type-2b ␥-aminobutyric acid receptor GABAb-R2 was found to be essential for the cell surface expression and the function of the GABAb-R1 subtype (7-9), suggesting that heterodimerization between the two receptor molecules is required for function. Biochemically, coimmunoprecipitation of receptors bearing different epitope tags was used to support the notion that GPCR homo-(10 -13) and heterodimers (9, 14) can form. However, the cons...

No abstract

The calcitonin receptor-like receptor (CRLR) is a seven-transmembrane domain (7TM) protein that requires the receptor activity-modifying protein 1 (RAMP1) to be expressed at the cell surface as a functional calcitonin gene-related peptide (CGRP) receptor. Although dimerization between the two molecules is well established, very little is known concerning the trafficking of this heterodimer upon receptor activation. Also, the subcellular localization and biochemical state of this ubiquitously expressed protein, in the absence of CRLR, remains poorly characterized. Here we report that when expressed alone RAMP1 is retained inside the cells where it is found in the endoplasmic reticulum and the Golgi predominantly as a disulfide-linked homodimer. In contrast, when expressed with CRLR, it is targeted to the cell surface as a 1:1 heterodimer with the 7TM protein. Although heterodimer formation does not involve intermolecular disulfide bonds, RAMP-CRLR association promotes the formation of intramolecular disulfide bonds within RAMP1. CGRP binding and receptor activation lead to the phosphorylation of CRLR and the internalization of the receptor as a stable complex. The internalization was found to be both dynamin-and ␤-arrestin-dependent, indicating that the formation of a ternary complex between CRLR, RAMP1, and ␤-arrestin leads to clathrin-coated pit-mediated endocytosis. These results therefore indicate that although atypical by its heterodimeric composition and its targeting to the plasma membrane, the CGRP receptor shares endocytotic mechanisms that are common to most classical 7TM receptors.The calcitonin gene-related peptide (CGRP) 1 is a member of the calcitonin family of regulatory peptides (1-3). Although a cDNA encoding the calcitonin receptor was cloned over 10 years ago (4), it was only recently that McLatchie et al. (5) established that co-expression of an orphan calcitonin-receptor like receptor (CRLR) with a newly discovered protein named receptor activity-modifying protein 1 (RAMP1) created a CGRP receptor. CRLR shares 55% amino acid sequence identity with the calcitonin receptor. Both belong to the family B of seventransmembrane domain (7TM) receptor. The family also includes the receptors for parathyroid hormone, parathyroid hormone-related protein, secretin, glucagon, and vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide (6, 7). RAMP1, for its part, is a member of a family that comprises 3 members designed RAMP1, RAMP2, and RAMP3. These small intrinsic membrane proteins share less than 30% of sequence identity and possess a large extracellular N terminus (ϳ100 amino acids), a single transmembrane domain, and a very short intracellular domain (10 amino acids) (5). One of the roles proposed for these proteins is that of chaperone/escort promoting the cell surface targeting of CRLR (5, 8). Interestingly, like CRLR, RAMPs are also retained intracellularly when expressed alone suggesting that an interaction between the two proteins is essential to their common cell...

The receptor activity-modifying proteins (RAMPs) and the calcitonin receptor-like receptor (CRLR) are both required to generate adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors. A mature, fully glycosylated, form of CRLR was associated with 125 I-CGRP binding, upon co-expression of RAMP1 and CRLR. In contrast, RAMP2 and -3 promoted the expression of smaller, core-glycosylated, CRLR forms, which were linked to AM receptor pharmacology. Since core glycosylation is classically a trademark of immature proteins, we tested the hypothesis that the coreglycosylated CRLR forms the AM receptor. Although significant amounts of core-glycosylated CRLR were produced upon co-expression with RAMP2 or -3, crosslinking experiments revealed that 125 I-AM only bound to the fully glycosylated forms. Similarly, 125 I-CGRP selectively recognized the mature CRLR species upon co-expression with RAMP1, indicating that the glycosylation does not determine ligand-binding selectivity. Our results also show that the three RAMPs lie close to the peptide binding pocket within the CRLR-RAMP heterodimers, since 125 I-AM and 125 I-CGRP were incorporated in RAMP2, -3, and -1, respectively. Cross-linking also stabilized the peptide-CRLR-RAMP ternary complexes, with the expected ligand selectivity, indicating that the fully processed heterodimers represent the functional receptors. Overall, the data indicate that direct protein-protein interactions dictate the pharmacological properties of the CRLR-RAMP complexes.In 1998, McLatchie et al. (1) reported that co-expression of two classes of polypeptides, a seven-transmembrane receptor known as the calcitonin receptor-like receptor (CRLR) 1 and accessory proteins termed receptor activity-modifying proteins (RAMPs), are required to generate functional receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). The formation of heterodimers between RAMPs and CRLR was found to be essential for the proper cell surface targeting and pharmacological characteristics of both CGRP and AM receptors (2, 3). The RAMP family comprises three members (RAMP1, -2, and -3) that share less than 30% sequence identity but a common topological organization. They are small intrinsic membrane proteins (predicted sizes: M r 14,000 -17,000) with a large extracellular N terminus (ϳ100 amino acids), a single transmembrane domain, and a very short intracellular domain (10 amino acids). Co-expression of RAMP1 with CRLR led to the formation of a CGRP receptor, whereas RAMP2 and -3 promoted the expression of an AM receptor (1).The distinct ligand-binding specificity imposed by the RAMP expressed with CRLR was accompanied by apparent differences in the receptor maturation pathways. The major CRLR species obtained upon co-expression with RAMP1 corresponded to a mature glycoprotein of M r 66,000 containing fully processed complex oligosaccharides. This species was associated with the binding of 125 I-h␣CGRP to the cell surface. In contrast, the major CRLR product observed with RAMP2 and -3 was a...