Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide.

The human beta 2 adrenergic receptor is a type IIIb membrane protein. It has a putative seven-transmembrane topology but lacks an amino-terminal cleavable signal sequence. The mechanism by which the amino terminus of the beta 2 receptor is translocated across the endoplasmic reticulum membrane is unknown. Furthermore, it is not known if translocation as a type IIIb protein is essential for the proper folding. Our studies indicate that conversion of beta 2 receptor from a type IIIb to a type IIIa membrane protein by introducing an NH2-terminal cleavable signal sequence enhances translocation of the receptor into the endoplasmic reticulum membrane, thereby facilitating expression of functional receptor.

The human B2 adrenergic receptor is a type IIIb membrane protein. It has a putative seven-transmembrane topology but lacks an amino-terminal cleavable signal sequence. The mechanism by which the amino terminus of the B2 receptor is translocated across the endoplasmic reticulum membrane is unknown. Furthermore, it is not known if translocation as a type IIIb protein is essential for the proper folding. Our studies indicate that conversion of B2 receptor from a type IIIb to a type IIIa membrane protein by introducing an NHz-terminal cleavable signal sequence enhances translocation of the receptor into the endoplasmic reticulum membrane, thereby facilitating expression of functional receptor.
The p2 adrenergic receptor is one of the most extensively characterized members of the family of G protein-coupled receptors, but little is known about its biosynthesis. Based on hydropathy analysis of the amino acid sequence and structural similarity to bacteriorhodopsin, it has been proposed that the seven hydrophobic segments of the & adrenergic receptor are membrane-spanning domains, and that the amino terminus of the receptor is on the extracellular side and the carboxyl terminus is on the cytoplasmic side (Dixon et al., 1986). This model has been supported by biochemical and immunocytochemical studies (Dolhman et aL, 1987;Wang et at., 1989).
Integral membrane proteins can be categorized into several different types, depending on the topological features such as the number of transmembrane segments and the orientation of the amino and carboxyl termini. The p2 adrenergic receptor, and most G protein-coupled receptors, can be classified as type IIIb proteins because they have a multiple membranespanning topology with their amino terminus on the extracellular side of the membrane but lack a cleavable signal sequence (Singer, 1990  amino acid residues with a basic, polar amino terminus and an apolar core domain (von Heijne, 1983). Signal sequences serve to direct the insertion of proteins into the membrane cotranslationally by a mechanism involving the signal recognition particle (Singer, 1990;Walter et al., 1984). Once the protein is inserted, the signal peptide is cleaved from the mature protein at the carboxyl-terminal extent of the signal sequence by signal peptidase present in the lumen of the endoplasmic reticulum (ER).' The mechanism of translocation of the amino terminus and first membrane-spanning domain of type IIIb proteins, such as the p2 receptor, is poorly understood. It is believed that the membrane insertion of proteins without a cleavable signal sequence is assisted by internal signal sequences which are inserted in the membrane by the same mechanism that operates for cleavable signal sequences, except that no postinsertional proteolysis occurs (Blobel, 1982;Sabatini et al., 1982;Wickner and Lodish, 1985;Spiess and Lodish, 1986). The hydrophobic, putative membrane-spanning domains of the p2 receptor are structurally most similar to known signal sequences. We sought to study the functional significance of the distinction between type IIIa and IIIb proteins, and to determine if a possible difference in the mechanism of translocation of a type IIIb protein compared to a type IIIa protein might be important in the proper folding of G protein-coupled receptors. A cleavable signal sequence from influenza hemaglutinin was added to the amino terminus of the human & adrenergic receptor. Receptors were expressed in insect cells and in a cell-free expression system. We observed that converting the p 2 receptor from a type IIIb to a type IIIa protein enhances translocation of the receptor into the ER membrane and production of functional protein.

EXPERIMENTAL PROCEDURES
Construction ofSFP2-The wild-type human P2 receptor cDNA was cloned into the transcription vector pSP65 (Promega, Madison, WI). To create SF&, a synthetic oligonucleotide of 76 base pairs was inserted in the NcoI site at the beginning of the P2 receptor coding sequence (Fig. 1). This synthetic oligonucleotide encodes a modified influenza hemaglutinin signal sequence (Jou et al., 1980) and an antigenic epitope of 8 amino acid residues (the "Flag" epitope, IBI, New Haven, CT). The "Flag" epitope was inserted after the cleavage site for signal peptidase in order to verify the cleavage of the signal p2 Receptor Expression sequence following its translocation. This epitope is recognized by a monoclonal antiserum (M1 anti-Flag antibody, IBI) only if there is no amino acid sequence preceding it (Prickett et al., 1989). Thus, the M1 antibody binds to the receptor only if the signal sequence is cleaved.
In Vitro Translation-The p2 and SFP, receptors were synthesized in a cell-free translation system using rabbit reticulocyte lysate and microsomal membranes from Xenopus oocytes as previously described (Kobilka, 1990). Translation was carried out in the presence of [35S] methionine for 30 min, and then the reaction was chased by adding 1 mM unlabeled methionine for an additional 90 min. Ligand binding studies on the translated receptor were carried out as described below. Radiolabeled proteins were analyzed by electrophoresis on a 10% polyacrylamide gel (SDS-PAGE gel) (Laemmli, 1970). The radioactivity of protein bands on a SDS-PAGE gel was quantified by using an AMBIS p scanner (AMBIS, San Diego, CA).
Expression of p2 Receptors in Insect (Sf9) Cells-DNA sequences encoding the wild-type p 2 receptor and SFD2 were cloned into the baculovirus expression vector pv11392 (provided by M. D. Summers, The calcium phosphate precipitation method was used to transfect plasmid and viral DNA into insect cells (Summers and Smith, 1987). The recombinant viruses containing 0 2 or SF& genes were isolated, plaque-purified, and titrated as previously described (Summers and Smith, 1987 At the indicated time points after infection, Sf9 cells were harvested by centrifugation at 200 X g at 4 "C. The cell pellets were resuspended in 1 ml of phosphate-buffered saline containing 10 nM [3H]DHA with or without other ligands. Reactions were incubated at 25 "C for 1 h. For binding studies on in vitro translated receptors, aliquots (4 &binding tube) of translation reaction mixture were incubated in 500 pl of binding buffer with 1 mg/ml bovine serum albumin and 100 PM '251-CYP for 90 min at 25 "C. The binding assays were terminated by rapid filtration over GF/C glass fiber filters. Filters were washed twice with 4 ml of ice-cold buffer, and radioactivity was quantified in a liquid scintillation counter for [3H]DHA and 35S-labeled compounds or in a y counter for '251-CYP. Nonspecific binding was defined by including 1O"j M (-)alprenolo1 in the reaction. Protein concentration was determined by the method of Bradford (1976).

RESULTS
Expression of p2 and SFpz in Insect Cells-To determine the effect of the signal sequence on receptor expression in intact cells, DNA sequences encoding p2 and SF@, were inserted into the identical site of a baculovirus expression vector. Plaque purified stocks of virus encoding each receptor were prepared, and the titer was determined. Cells were infected with four equivalent dilutions from equal titer virus stocks carrying either pz or SFP, receptor genes. Receptor expression was assayed by [3H]DHA binding at 24, 36, 48, and 96 h after infection. For each viral dilution and at each corresponding time point, the level of functional receptor produced (expressed as cpm specific binding/mg protein) was approximately %fold greater for SF@, compared with p2 receptors ( Fig. 2 and Table I).
The pharmacological properties of SFP, receptors are indistinguishable from those of wild-type p2 receptors expressed in Sf9 cells. Competition studies were performed using two agonists (isoproterenol and epinephrine) and two antagonists (propranolol and alprenolol) against [3H]DHA binding. The curves for p2 and SF& are essentially identical (Fig. 3) A total of three independent experiments were performed. See Table  I  Solubilized SF& produced in insect cells bound efficiently to an M1 antibody column (data not shown). As indicated under "Experimental Procedures," the Ca2+-dependent M1 antibody recognizes the Flag epitope only when nothing precedes the epitope at the amino terminus. Thus, the signal peptide is cleaved in insect cells.
Cell-free Expression of SFp2-To explore the mechanism by which the signal peptide enhances the expression of functional receptor in insect cells, we studied expression of SF@, and wild-type pz in a cell-free expression system composed of rabbit reticulocyte lysate and membranes prepared from Xenopus laevis oocytes. We have previously shown that receptor expressed in this cell-free translation system is capable of ligand binding and exhibits the same pharmacologic properties as receptors expressed in cultured cells (Kobilka, 1990).
The mRNA encoding SF& and wild-type pZ receptors was translated in the presence of [35S]methionine for 2 h at 30 "C. The translation mixture was analyzed for ligand binding and incorporation of [35S]methionine-labeled protein into membranes. Maximal specific '251-CYP binding normalized to "Slabeled membrane protein was determined for p2 and SF&.
As can be seen in Table 11, part A, translation of SFp, resulted in 2-4-fold more '2sII-CYP binding/cpm total 35S-labeled mem-  Receptor proteins were synthesized in the presence of ["S]methimine: a small aliquot of the translation reaction was analyzed on a 10% SDS-PAGE gel, and the rest was used for radioligand binding in the presence of 100 p~ ""I-CYP as described under "Experimental Procedures." A, specific binding normalized to total membrane-associated ""S-labeled receptor protein: cpm for specific "'I-CYP hinding divided by cpm for '%-labeled membrane protein trapped on the litters for binding assays and counted in a Reckman liquid scintillation counter. R, specific binding normalized to fully glycosylated protein: cpm for '"I-CYP binding divided by cpm for :%labeled fully glycosylated, 52-kDa protein measured with the AMBIS scanner from the dried 10% polyacrylamide gel. Radioactivity for ""I-CYP binding was quantified on a Reckman y counter. The cpm values obtained by liquid scintillation and AMBIS scanner are not directly comparable 1)ecause of the different counting efficiencies. Two independent experiments were carried out, and the individual values for each experiment are listed below. brane protein than translation of &. The difference in expression between SFP, and P2 in the cell-free expression system is therefore similar to the difference observed in the insect cell expression system. T o study the influence of the signal peptide on translocation of the receptor, the products of translation were analyzed by SDS-PAGE. Fig. 4 shows the result of translating mRNA  (Fig. 4, lane 3 ) , the translated receptor protein migrates as a 42-kDa band on a SDS-PAGE gel. When membranes are added to the translation reaction, at least two additional bands with larger molecular mass (44 and 52 kDa) are apparent (Fig. 4, lunes 4 and 6). These slowly migrating species are receptor proteins that are modified by asparagine-linked glycosylation because they are not observed when translation is carried out in the presence of a competitive glycosylation inhibitor Asn-Tyr-Thr (NYT) (Lau et al., 1983) (Fig. 4, lune S). 1 and 2). The human P2 receptor has three consensus sites for N-linked glycosylation within putative extracellular domains: two in the amino terminus and one in the hydrophilic sequence between the fourth and fifth hydrophobic domains. Since glycosylation occurs only in the lumen of the endoplasmic reticulum, partial glycosylation of receptor protein may indicate that one of the domains containing consensus sites for N-linked glycosylation has not been translocated. In comparison to the wild-type receptor, the amount of partially or non-glycosylated proteins are clearly reduced in SFP, (Fig.   5, lane 1 versus 3 )  suggest that: I ) a significant portion of the wild-type Pz is incompletely translocated (as evidence by partial glycosylation) and that some or all of this partially translocated protein is nonfunctional; and 2) the signal sequence improves expression of the P2 receptor by enhancing translocation of the amino terminus. Fig. 6 shows that the fully glycosylated form of detergent-solubilized SFPz binds to the M I antibody, indicating that the signal peptide has been cleaved to unmask the Flag epitope.

DISCUSSION
In the present study, we have examined the effect of converting the l j 2 receptor from a type IIIb membrane protein to a type IIIa membrane protein. The amino-terminal hydrophobic segment of a type IIIb protein is believed to serve as its own noncleavable signal sequence (Rlobel, 1980;Singer, 1990). Of interest, cleavable sequences are believed to be inserted into the ER membrane with the amino terminus on the cytoplasmic side of the membrane. In contrast, the first membrane-spanning domain of G protein-coupled receptors is believed to be oriented with the amino terminus on the luminal side of the membrane. We sought to determine whether the addition of a cleavable signal sequence would alter receptor biosynthesis. Our results show that when the amount of functional protein (as assessed by ligand binding) is normalized to total membrane-associated protein (%labeled protein trapped by filtration through a glass fiber filter), translation of mRNA encoding SF& produces approximately 2-fold more functional protein than the wild-type &. This is comparable with the results obtained by expression of these receptors in insect cells (Fig. 2). The enhancement in expression of functional receptors that results from the addition of a signal peptide appears to be due to more efficient translocation of the modified receptor into the ER membrane. Fig. 5 shows that, in comparison to the wild type /& receptor, R greater proportion of translated SF& is fully glycosylated. While we have previously shown that the presence of Nlinked oligosaccharides on the p2 receptor is not required for function, glycosylation is nevertheless a reliable indicator that the glycosylated domain has been translocated into the lumen of the ER. Our results indicate that the mechanism by which the amino terminus of the p, receptor is translocated as a type IIIb membrane protein appears to he less efficient than the mechanism employed by type IIIa proteins. The increase in expression of functional protein afforded by the signal sequence would not be expected to have physiologic significance, yet it remains to be explained why this class of membrane proteins has evolved with a less efficient mechanism for translocation.