The avian beta-adrenergic receptor: primary structure and membrane topology.

Abstract

Partial amino acid sequence information allowed the isolation of cDNA clones encoding the turkey erythrocyte (3-adrenergic receptor. Antisera raised against synthetic peptides encoded by the cDNA crossreacted with the purified receptor and appropriate tryptic fragments, confirming the identity of the cDNA. The receptor is composed of 483 amino acids and has a molecular mass of 54 kDa. Its sequence suggests that it is arranged predominantly in seven membranespanning sequences and a long cytoplasmic carboxyl-terminal domain. The extracellular amino-terminal domain contains a consensus sequence for N-glycosylation. The /3-adrenergic receptor displays overall structural similarity and weak sequence homology with rhodopsin. Because both proteins act by regulating GTP-binding proteins, a compact structure based on seven membrane-spanning regions may be a general model for receptors that act on G proteins. (1). Functionally, the receptor is phylogenetically conserved. The receptor purified from turkey erythrocytes can efficiently regulate GO from rabbit liver in reconstituted phospholipid vesicles (3, 4), and its selectivity for numerous agonist and antagonist ligands is only slightly discrepant from the mammalian 31-adrenergic receptor (5).Whether the receptor interacts with G. on the hydrophilic cytoplasmic face of the plasma membrane or within the bilayer is unknown; nor is anything known about the structural details of this regulatory interaction. Presumably, all receptors that activate G proteins will share this regulatory domain, and definable differences will exist in the homologous regions of receptors that activate different G proteins.The sites of P-adrenergic ligand binding, regulatory phosphorylation, and stimulatory reduction by thiols (6) are also of great interest. Much of the difficulty in learning about the structure of the f3-adrenergic receptor is due to its low concentration in plasma membranes: P-adrenergic receptor must be purified over 20,000-fold from an already wellpurified plasma membrane fraction. We have therefore undertaken the cloning of the cDNA that encodes the Padrenergic receptor as a first step toward more direct studies of its structure and function. The sequence of the 82-adrenergic receptor from hamster lung has appeared recently (7), and homology between the two sequences and the sequence of rhodopsin suggests functionally important aspects of their structures. METHODSP3-Adrenergic receptor was purified from turkey erythrocyte plasma membrane as described by Brandt and Ross (4). This preparation, which consists mainly of an active 40-kDa proteolysis product and the 53-kDa receptor (1, 4), was separated from digitonin and minor impurities by HPLC on a 300-A pore size, C4 column (Synchrom, Linden, IN) using a linear gradient of0.1% trifluoroacetic acid in water to 0.06% trifluoroacetic acid in 50% 1-propanol (vol/vol). The receptor was cleaved with cyanogen bromide, 60 mg/ml in 70% (vol/vol) formic acid, for 24 hr at room temperature under nitrogen. Sequencing was performed...

Chemical characterization of ligand binding site fragments from turkey β‐adrenergic receptor

“…Incubation of the affinity-labeled 50 kDa receptor with endoglycosidase F resulted in a change in its electrophoretic migration corresponding to a molecular mass of 45 kDa (not shown), as described before [12], indicating that the carbohydrate moiety located in the N-terminal region [2,7] of the affinity-labeled t45'AR was removed by the glycosidase. Upon treatment of the NTCBlabeled fragments (FI8, F23, F28) with endoglycosidase F, an electrophoretic pattern identical to that of the untreated fragments ( fig.…”

“…residues located along the polypeptide chain [2]; indeed, the smallest labeled polypeptide observed after cleavage was still twice the size (15 kDa) of the longest segment between two neighbouring cysteine residues present in the protein (Cysl°-CysSS).…”

“…Based on this knowledge, we assumed that the cysteine residues present in the transmembrane domains of the tDAR [2] were not cleaved by NTCB. Since F18, F23 and F28 do not contain the carbohydrate moiety of t45'AR, they should be located between Cys H4 and the Cterminal end.…”

“…F37 and F21, obtained following NTCB cleavage of intact labeled membranes, are proposed to have Tyr 437 as their C-terminal and Cys H4 (or Cys ll6) and Cys TM as their respective N-terminals. Arrows indicate proposed cleavage sites of cysteine residues; numbers indicate the position in the primary structure; I-VII represent the transmembrane domains of the receptor [2]; (e) Cys residues; (o) Asp residue; (y) carbohydrate moiety.…”

“…The turkey Z?-adrenergic receptor (tflAR), isolated from erythrocyte plasma membranes, was one of the first receptors of this type for which the amino acid sequence was determined [2]. Although a putative model of its folding in the membrane was deduced from sequence homology and resemblance to the light receptor rhodopsin, the exact nature and location of the catecholamine- binding site require further investigation.…”