Identification, Sequence, and Expression of Caveolin-2 Defines a Caveolin Gene Family PHILIPP E

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 131-135, January 1996 Cell Biology

Identification, sequence, and expression of caveolin-2 defines a caveolin gene family PHILIPP E. SCHERER*, TAKASHI OKAMOTOt, MIYOUNG CHUN*, IKUO NISHIMOTOt, HARVEY F. LODISH*§, AND MICHAEL P. LISANTI*¶ *The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142-1479; tShriners Hospitals for Crippled Children, Massachusetts General Hospital, Department of Anesthesia, Harvard Medical School, Boston, MA 02114; tCardiovascular Research Center, Massachusetts General Hospital-East, Department of Medicine, 149 13th Street, Charlestown, MA t)2129; and §Department of Biology, Massachusetts Institute of Technology, Cambridge, MA t)2139 Contributed by Harvey F. Lodish, September 19, 1995

ABSTRACT Caveolin, a 21- to 24-kDa integral membrane that coexpression of a- and ,B-caveolin within a single cell may protein, is a principal component of caveolae membranes. be used to generate at least two distinct subpopulations of Caveolin interacts directly with heterotrimeric guanine nu- caveolae that may be differentially regulated by a specific cleotide binding proteins (G proteins) and can functionally caveolin-associated serine kinase (14). regulate their activity. Here, an 20-kDa caveolin-related protein, caveolin-2, was identified through microsequencing MATERIALS AND METHODS of adipocyte-derived caveolin-enriched membranes; caveolin was retermed caveolin-1. Caveolins 1 and 2 are similar in most Materials. Caveolin-rich membrane domains were purified respects. mRNAs for both caveolin-1 and caveolin-2 are most from murine adipose tissue by an established protocol (15), abundantly expressed in white adipose tissue and are induced except that purified adipocyte plasma membranes were used as during adipocyte differentiation. Caveolin-2 colocalizes with starting material (18). Internal microsequencing was per- caveolin-1, indicating that caveolin-2 also localizes to caveo- formed as described (4). A rabbit polyclonal antibody directed lae. However, caveolin-1 and caveolin-2 differ in their func- against the C-terminal 44 amino acids of caveolin-1 (residues tional interactions with heterotrimeric G proteins, possibly 135-178) has been characterized (14); it recognizes both a- and explaining why caveolin-1 and -2 are coexpressed within a ,B-isoforms of caveolin-1 but does not recognize caveolin-2. single cell. Northern Blot Analysis. 3T3-L1 mouse fibroblasts (ATCC CCL 92.1) were propagated and differentiated according to the conventional protocol (18). mRNA isolation from tissues Caveolae are plasma membrane specializations present in and 3T3-L1 cells at various stages of adipocyte differentiation most cell types (1). They are most conspicuous in adipocytes and agarose gel electrophoresis/transfer to nylon membranes were they represent up to 20% of the total plasma membrane was performed as described (18); filters were washed in 2x surface area (2). Cytoplasmically oriented signaling molecules SSC/0.1% SDS and 0.5x SSC/0.1% SDS at 50°C. are concentrated within these structures, including heterotri- Recombinant Expression and Selection of Stable Cell Lines. meric guanine nucleotide binding proteins (G proteins), Src- An epitope-tagged form of caveolin-2 was subcloned into the like kinases, protein kinase Ca and Ras-related GTPases multiple cloning site of the vector pCB7 (containing the (3-9). The caveolae signaling hypothesis states that caveolar hygroR marker; gift of J. Casanova, Massachusetts General localization of signaling molecules could provide a compart- Hospital). The myc-epitope tag was incorporated into the N mental basis for integrating certain transmembrane signaling terminus (MEQKLISEEDLNGG-caveolin-2) of the cloned events (1). Caveolin, a 21- to 24-kDa integral membrane human caveolin-2 cDNA using PCR primers. GG was placed protein, is the main component of caveolae membranes (10). as a spacer between the epitope and the caveolin-2 coding Structurally, caveolin can be divided into three distinct regions: sequences, as previously done for caveolin-1 (12, 14, 19). a hydrophilic cytosolic N-terminal domain, a membrane- DNAs were transiently transfected into COS-7 cells by the spanning region, and a hydrophilic C-terminal domain (11). DEAE-dextran method; 3T3-L1 fibroblasts were stably trans- The C-terminal domain undergoes palmitoylation (S- fected by a modification of the calcium phosphate precipita- acylation) on three cysteine residues (12), suggesting that both tion procedure (14). 3T3-L1 fibroblasts expressing recombi- the membrane-spanning region and the C-terminal domain of nant caveolin-2 were grown to confluence in 150-mm dishes caveolin are associated with the membrane. Recent evidence and used to purify caveolin-rich membrane domains essentially suggests that caveolin may function as a scaffolding protein for as described (3, 4, 15, 20, 21). Immunostaining was performed organizing and concentrating certain caveolin-interacting mol- as described (5), with minor modifications. ecules within caveolae membranes (3, 13). GTP Hydrolysis and GTP[yS] Binding Assays. Trimeric G,, Although caveolin is the product of a single gene, it encodes protein purified from bovine brain was kindly provided by T. one mRNA but two caveolin isoforms that differ by 3 kDa Haga (22). Steady-state GTP hydrolysis activity and GTP[yS] and have been termed a- and ,B-caveolin (14). a-caveolin binding were measured as described (23, 24). The caveolin-2- contains residues 1-178; methionine 32 acts as an internal derived polypeptide contained residues 54-73 of caveolin-2.1 translation initiation site to form ,B-caveolin (14). Both caveolin isoforms are targeted to caveolae (15), form homooli- RESULTS gomers (16, 17), and interact with G proteins (13). However, a- and ,B-caveolin assume a distinct but overlapping subcellular Identification of an 20-kDa Caveolin-Related Protein distribution in intact cells (14) and only ,B-caveolin is phos- That Is Expressed in Adipocytes and That Copurifies with phorylated on serine residues in vivo (15). These results suggest Abbreviations: G protein, guanine nucleotide binding protein; GDI, GDP dissociation inhibitor; GAP, GTP-ase activating protein. The publication costs of this article were defrayed in part by page charge ~To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in 'The sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U32114). 131 Downloaded by guest on September 24, 2021 132 Cell Biology: Scherer et al. Proc. Natl. Acad. Sci. USA 93 (1996)

Caveolin. To identify novel adipocyte caveolar components, nos. H21944, H01244, and R01546). These clones derive from caveolin-enriched membrane domains were purified from human cDNA libraries from diverse tissue sources (placenta, murine adipocyte plasma membranes using an established breast, and fetal liver/spleen), suggesting that many tissues protocol. The major protein components of these adipocyte- express low levels of caveolin-2. However, no reported EST derived domains were identified by microsequence analysis sequence contains the entire caveolin-2 coding sequence- (not shown). Many of these components represent known even when these EST sequences are assembled. proteins that were previously shown to copurify with caveolin Fig. 2A shows that like caveolin-1, caveolin-2 mRNA is most using lung parenchyma as the starting material (4, 14). A abundantly expressed in white adipose tissue, differentiated peptide sequence derived from an 20-kDa protein was 3T3-L1 adipocytes, and lung tissue. This is consistent with obtained through this analysis (KLGFEDXIAEPXXTH). This morphological evidence that differentiated 3T3-L1 adipocytes peptide is strikingly homologous to caveolin but differs in are a rich source of caveolae (2). Cultured 3T3-L1 fibroblasts several residues (denoted in boldface) from the known se- offer a convenient system to study adipocyte differentiation. quence of murine caveolin. As this region of caveolin is These cells can be induced to differentiate over 8 days from absolutely conserved from chicken to human (25), this peptide fibroblasts into adipocytes. Like caveolin-1, caveolin-2 mRNA could derive from a caveolin-related protein that copurifies is strongly induced on day 4 of differentiation (Fig. 2B). with caveolin-rich membrane domains. Data base searches Relative to the constant expression of hsp70 mRNA, caveo- revealed that this peptide is potentially encoded by an ex- lin-1 and caveolin-2 mRNAs are induced -25-fold. pressed sequence tag of 345 nucleotides (KLGFEDVIA- Recombinant Expression and Properties of Caveolin-2. To EPVTTH; residues differing from caveolin are in boldface; study the properties of caveolin-2, an epitope-tagged form of GenBank accession no. T84152). This human cDNA clone the protein was expressed in transfected cells. Epitope-tagging (clone 110467) was obtained from the Washington University- of caveolin-1 with the same epitope does not affect its behavior Merck Expressed Sequence Tag Project for further analysis. or its subcellular localization within caveolae membranes (12, Sequence and Tissue-Specific Expression of Caveolin-2, a 14, 19). Member of the Caveolin Gene Family. Analysis and sequenc- Caveolin-1 exists as an -350-kDa homooligomer containing ing of clone 110467 revealed that it contains an 1.4-kb insert 14-16 monomeric units per oligomer (16, 17). Caveolin-1 with an open reading frame encoding a caveolin-related homooligomers may function as docking sites to concentrate protein of 149 amino acids. For simplicity, caveolin was signaling molecules, such as heterotrimeric G proteins, within retermed caveolin-1 and this gene product was designated as caveolae membranes (16). Residues 61-101 of the N-terminal caveolin-2. Fig. 1 shows the deduced protein sequence of cytoplasmic domain of caveolin-1 are sufficient to mediate human caveolin-2 and its alignment with the known sequences these caveolin-caveolin interactions (16). As the correspond- of human caveolin-1 and caveolin-1 from other species. Hu- ing 41-amino acid region of caveolin-2 is 50% identical to man caveolin-2 is 38% identical and 58% similar to human caveolin-1 with half of the changes reflecting conservative caveolin-1. The most conserved region is a stretch of eight substitutions, we suspected that caveolin-2 might also form a identical amino acids (FEDVIAEP) within the N-terminal high molecular mass oligomer. To evaluate this possibility, the domains of caveolin-1 and caveolin-2. This may represent a same techniques were employed as used previously for caveo- signature sequence that is characteristic of members of the lin-1 (16). Homooligomers of caveolin-1 are resistant to dis- caveolin gene family. Both caveolin-1 and caveolin-2 contain a sociation by a mixture of SDS and 2-mercaptoethanol, except 33-amino acid membrane-spanning segment and a hydrophilic at elevated temperatures (boiling at 100°C) (16, 17). Thus, 43- to 44-amino acid C-terminal domain. The N-terminal these conditions were used to determine whether caveolin-2 domain of caveolin-2 is 26 amino acids shorter than caveolin- forms similar high molecular mass oligomers. As a positive la; caveolin-2 migrates on SDS/polyacrylamide gels with a control, the oligomeric state of N-terminally myc-tagged molecular mass of -20 kDa (see Fig. 3A), although it has a caveolin-1 was evaluated in parallel. Fig. 3A shows that caveo- predicted molecular mass of 16.8 kDa. Similarly, caveolin-la lin-2 migrated mainly as a monomer under the same conditions migrates at -24 kDa, although it has a predicted molecular that caveolin-1 migrated as a high molecular mass oligomer of mass of 20.5 kDa. -350 kDa (lanes 3 and 4). However, a minor '40-kDa species A search of existing data bases with the deduced caveolin-2 of caveolin-2 (arrow, lane 4) is consistent with a dimeric protein sequence reveals three other expressed sequence tags structure for caveolin-2. In further support of this notion, (ESTs) that should encode caveolin-2 (GenBank accession caveolin-2 migrated in velocity gradients between molecular * human. Cav-2 MD O D S 7 S HH S G L E Y E K - F A SDs 0------D R D P H R[L N human Cav-1 M S G G K IY V D S E G H LY T VP I R E S K PNN KAM A ELSE Y D LV N P K NI GNI y D K 0 V D A H T |R D H L dog CaOv-1 M S G G K V D S E G HL Y T V PI R E S K PNN KAM A EMSE Y V N PIK IYI GNI y E K Q V D A H - KEIKE D L R D H|L NI rot. Cav - 1 M S G G K V D S E G H L YI T V P R E S K PNN KAM A EVNE D L V N R NI IYI GNIGN y D K O V Y D A H T KE D P|K HIL mouse. Cav-1 M SG G K V D SE G H L Y T V R E S K PNN KAM A EVTE V D L V N R O NI IYI IP GNI y D K Q Y D A H T KEIKE P|K HIL chick Cov-i J S G T K S V O S E |£ F L IY 1 A LJ V R E a K PNN KMM A D ELSE K A V H D V D T D L V N [R D P K H LL NJ

human Cav-2 34 S H L - K L G F E D V I A E P V T T H S FD K VW I C SH A LF E I S KYV M K F L T V F L A IP LA F A GI L F A human. Cav-1 61 O D V V K D F E D V I A E P E G T H S FEG I W K A S FT T F T E T K Y W F Y R LL S A L FG I P M A L I W G I Y F A dog. Cav-1 61 DOV VIKI F E DV I AE P EG TH S FOG WK ASF TT F T VT KYWF YR LL SA L FG I P MAL IW G Y FA rat .Cav-1 61 D D V V K ID F E D Y I A E P EG T H S F O G W K A S F TT F T V T K Y W F Y R L L S T ! F G I PM A L I W G I Y mouse. Cov-1 61 D D I AE P E G T H SF DG I W K A S F T T F T V T K Y W F Y R L L S T I F G I P M A L I W G I Y VVK DFEDV chick Cov-1 61 LKVVIJ F E DV IA EP.E G TH SF DG WJKA8F T TLFjTV TKYGYLL A PIPYA L IWIYFW

humor Cov-2 93 T L C LHIWI L MI F V KT C M V L P S V O T I W K S V T D V I A P L C T S VR C FSSV S L r0 S Q D human. Cov-1 121 L SIFL H I WIA IPIV C ! K|S F L E O C T S R V Y S ! Y V H TV CDIP L F E AIV GIK I FF S R I NLJ0 K E dog. Cav-1 121 L S F L H I WA VIPI C K S F L E S C S R V Y S I Y V HT F C D P F F E AV G K F S N R I N M O K E T rat. Cv -1 121 L S F L H I W A V V IPI C K S F L E S C S R V Y S I Y V H NFC OIP L F G G GK F S N P S T O E E I mouse Cav-1 121 L S F L H I WA VV IPI C KS F L E S C S R V Y S YV H TFCD PL FE A IGKI F S N R ! S T 0 K E I chick. Cav-1 121 IL S| F|L H I W A V V PJC R S Y LJ: E I 0 C SR V Y S C H T F C D PL FE A M K V FS R A T V R K E I

FIG. 1. Deduced protein sequence of human caveolin-2. Protein sequence of human caveolin-2 is compared with the known sequences of human, dog, rat, mouse, and chicken caveolin-1. Residues identical to caveolin-2 are boxed. Position of the G-protein binding region of caveolin-1 is overlined; position of the hydrophobic membrane spanning region is indicated by a boldface overline. A conserved methionine residue (an alternate translation initiation site for generating caveolin-1,B) is indicated by an asterisk. Note that both caveolin-1 and caveolin-2 have the same overall structure with a 33-amino acid hydrophobic region. Downloaded by guest on September 24, 2021 Cell Biology: Scherer et al. Proc. Natl. Acad. Sci. USA 93 (1996) 133 A B 0cO4) 9;b2 ;6 - .(~~~~.~ o~01 V" V Vl INN,04e4 9.4- 9.4- 7.5- 7.5- 4.4- 4.4- 2.4- - .' 2.4- Cav-i 0 Cav-1 am 1.4- 1.4- 0.24. 0.24-

9.4- 9.4- 7.5. 7.5- 4.4- 4.4-

2.4. 9_ 4I Cav-2 2.4- . 40 464* ~Cav-2 1.4- 1.4- 0.24- 0.24- s .*.

_ .a F '-''' Hsp7O . .S am_ ,,~b Hsp7O

FIG. 2. Caveolin-2 mRNA is most abundant in adipose tissue and is induced during differentiation of 3T3-L1 cells to the adipocyte form. (A) Northern blot of RNA from mouse tissues. Poly(A)+ RNA (0.5 ,ug) harvested from different tissues was separated on a 1% agarose gel containing formamide and transferred to a nylon membrane. Blots were first probed with a fragment containing the entire coding sequence of caveolin-2, stripped, and subsequently reprobed with the cDNAs for caveolin-1 and cytosolic hsp7O (as a control for equal loading). 3T3-L1 diff, mRNA isolated from day 8 3T3-L1 adipocytes. Numbers at left indicate molecular mass standards (in kb). (B) Northern blot of RNA from differentiating 3T3-L1 cells. Poly(A)+ RNA (0.5 jig), harvested from cells on different days after induction of 3T3-L1 fibroblasts to adipocytes, was analyzed as described in A. Numbers at left indicate molecular mass standards (in kb). A ,>- 5v N~Y CIV B A c2 tO 1 2 3 4 5 6 7 8 9 10 11 12 13 00 _ 97 68 200- __z -43 29 44 'r- 92- Ponceau S -18 68- 1 1

45-

c 29- Cav-i 21- 14- Cav-2 1 2 3 4 FIG. 3. Recombinant expression of epitope-tagged caveolin-2. (A) Detection of SDS-resistant caveolin homooligomers. COS-7 cells transfected with N-terminally myc-tagged caveolin-1 or caveolin-2 were lysed in sample buffer and run on a 5-12% acrylamide gradient gel. Caveolin-1 and caveolin-2 were visualized after transfer to nitrocellulose by immunoblotting with monoclonal antibody 9E10 that recognizes the myc epitope. Lanes 1 and 2, heat-treated prior to loading (boiling at 100°C for 2 min); lanes 3-4, without boiling. Expression of caveolin-2 in COS-7 cells yielded a protein product of the expected molecular mass (18-20 kDa; lane 2) that is slightly smaller than caveolin-1 (lane 1). Note that heat-dissociated caveolin-1 migrated as a monomer (lane 1), while undissociated caveolin-1 migrated predominantly as a high molecular mass oligomer of 350 kDa (lane 3), as we have shown previously (16). In contrast, caveolin-2 migrated as a monomer under both conditions (lanes 2 and 4). However, a dimeric form of caveolin-2 was also observed (lower arrow, lane 4). (B) Subcellular fractionation of 3T3-L1 fibroblasts stably expressing myc-tagged caveolin-2. Distribution of total cellular proteins, caveolin-1, and caveolin-2 is shown. One-milliliter sucrose gradient fractions were collected from the top and analyzed by Ponceau S staining (Top) or immunoblotting (Middle and Bottom). Fractions 1-8 are the 5-30% sucrose layer, fractions 9-12 are the 40% sucrose layer, and fraction 13 is the insoluble pellet. Fractions 9-12 represent the loading zone of these bottom-loaded flotation gradients and contain the bulk of cellular membranes and cytosolic proteins. Note that fractions 5 and 6 retain >95% of caveolin-1 and caveolin-2 and exclude most cellular proteins and markers for endoplasmic reticulum, Golgi, noncaveolar plasma membrane, mitochondria, and lysosomes, as we and others have shown (3, 4, 15). Immunoblotting was performed with anti-caveolin-1 IgG (monoclonal antibody 2297; 1:400) or with 9E10 ascites fluid (1:500) to visualize myc-tagged caveolin-2. Downloaded by guest on September 24, 2021 134 Cell Biology: Scherer et al. Proc. Natl. Acad. Sci. USA 93 (1996) mass standards of 29 and 66 kDa (not shown). However, caveolin-2 may form stable high molecular mass oligomers under other conditions. To determine whether caveolin-1 and caveolin-2 colocalize, 200 caveolin-2 was stably expressed in 3T3-L1 fibroblasts, which -D express endogenous caveolin-1. A protocol involving homog- enization in Triton X-100 followed by equilibrium sucrose CD) density centrifugation was used to separate membranes enriched in caveolin-1 from the bulk of cellular membranes and cytosolic proteins (3, 4, 8, 13-15, 20, 21, 26). In this fractionation scheme, immunoblotting with anti-caveolin-1 IgG can be 1 50 used to track the position of caveolae-derived membranes. Fig. 3B illustrates that 90-95% of both caveolin-1 and caveolin-2 cofractionate in the same low density fractions, indicating that 0 they colocalize. 0C0.1 3 5-1 Immunostaining of 3T3-L1 fibroblasts expressing myc- -o tagged caveolin-2 revealed punctate fluorescence or micro- patches along the cell surface (Fig. 4B, solid arrows) and within 0~ the perinuclear region (open arrow). This staining pattern is reminiscent of that obtained previously for caveolin-1 (3, 5, 10, 14). Indeed, double labeling of these cells with specific antibody probes that distinguish between caveolin-1 (C-terminal- specific anti-rabbit IgG) and caveolin-2 (9E10; anti-myc mono- 0 0.1 1 3 5 10 clonal antibody) revealed significant colocalization of these Peptide concentration (igM) two distinct gene products at the cell surface (compare Fig. 4 FIG. 5. Effect of the caveolin-2-derived polypeptide on the func- A and B). tional activity of purified heterotrimeric G proteins. (A) The effect of A Caveolin-2-Derived Polypeptide Activates the GTPase the caveolin-2-derived peptide on GTPase activity of purified trimeric Activity of Purified Heterotrimeric G Proteins. Caveolin-1 Go protein. Activity is expressed as a percentage of the basal activity, functionally interacts with multiple heterotrimeric G proteins which was 0.17 ± 0.01 min-1 (n = 3; mean ± SE). (B) Effect of the (13); residues 82-101 of caveolin-1 mediate the interaction peptide on GTP[yS] binding activity of purified trimeric Go protein. Activity is expressed as percentage of basal activity, of which the Kapp was 0.20 ± 0.02 min 1 (n = 3; mean ± SE). All experiments were done independently at least three times, and values indicate means ± SE. between caveolin-1 and G-protein a subunits. In addition, a caveolin-1-derived peptide encoding residues 82-101 can functionally suppress the basal GTPase activity of purified heterotrimeric G proteins, acting as a GDI (GDP dissociation inhibitor) (13). The corresponding region of caveolin-2 is -30% identical to caveolin-1, with almost half of these changes representing conservative substitutions (Fig. 1). Thus, a peptide encoding this region of caveolin-2 was generated to evaluate its effect on the basal activity of purified heterotrimeric G proteins. Fig. SA shows the effect of this caveolin-2-derived polypeptide on the GTPase activity and GTP[-yS] binding of purified trimeric Go protein. Unlike polypeptides derived from caveolin-1, micromolar concentrations of this caveolin-2-derived polypeptide stimulated the GTPase activity of trimeric Go protein with an EC50 value of 5 p,M. At 10 ,tM, this activation corresponded to >200% of the basal activity of Go protein. In contrast, this polypeptide had no significant effect on the binding of GTP[yS], even at 10 ,uM (Fig. 5B). Thus, this caveolin-2-derived polypeptide behaves differently than the corresponding peptide from caveolin-1, as this caveolin-2 FIG. 4. Immunolocalization of caveolin-1 and caveolin-2 within a polypeptide appears to encode a GAP (GTPase-activating single cell. Double immunofluorescence labeling of caveolin-1 and protein)-like activity. However, GDI and GAP activities could myc-tagged caveolin-2. Caveolin-2 was stably expressed in 3T3-L1 act in concert, as both would serve to hold (GDI or fibroblasts, which contain endogenous caveolin-1. These cells were activity) fixed, permeabilized, and subjected to sequential incubations with actively place (GAP activity) G, subunits in the inactive specific antibody probes. (A) Caveolin-1 expression detected with a GDP-bound conformation. rabbit polyclonal IgG probe that specifically recognizes only caveolin-1. (B) Caveolin-2 expression detected with mAb 9E10 that recognizes the myc-epitope. Control experiments with singly transfected DISCUSSION populations of COS-7 cells confirmed the specificity of these antibody Here we show that caveolin is a member of a newly discovered probes; no cross-reaction was observed. Solid arrows point at regions gene family of related molecules. Thus, caveolin was retermed of cell surface staining and open arrows point at regions of perinuclear staining. Bound primary antibodies were visualized by incubation with caveolin-1 and the new member of this family was designated distinctly tagged fluorescent secondary antibodies (Cy-3-conjugated as caveolin-2. This represents a second mechanism for gener- for caveolin-1 and fluorescein-conjugated for caveolin-2) as described. ating caveolin diversity: while there are two isoforms of (Bar = 5 ,um.) caveolin-1, they derive from a single caveolin gene (14). Downloaded by guest on September 24, 2021 Cell Biology: Scherer et al. Proc. Natl. Acad. Sci. USA 93 (1996) 135 Caveolin-2 was identified through microsequencing of Institutes of Health grant (GM-50443) to M.P.L., National Institutes caveolin-rich domains purified from adipocyte plasma mem- of Health grants (GM-49516/DK-47618) to H.F.L., and a grant from branes, indicating that caveolin-1 and caveolin-2 are coex- Bristol-Myers Squibb to I.N. P.E.S. is funded by a Swiss National pressed within adipocytes (most likely within the same cell). Science Foundation fellowship; T.O. was the recipient of fellowships Caveolin-2 copurifies with caveolin-1, suggesting their colo- from the Byotai-Taisha Foundation and the Mochida Memorial calization within caveolae membranes. In support of this Foundation; M.C. was supported by a fellowship from the Life conclusion, caveolin-1 and caveolin-2 have the same tissue Sciences Research Foundation. distribution, are most abundantly expressed in white adipose 1. Lisanti, M. P., Scherer, P., Tang, Z.-L. & Sargiacomo, M. (1994) tissue, and are dramatically induced during adipocyte differ- Trends Cell Biol. 4, 231-235. entiation. These results indicate that both caveolin family 2. Fan, J. Y., Carpentier, J.-L., van Obberghen, E., Grunfeld, C., members are important for adipocyte function. Caveolae are Gorden, P. & Orci, L. (1983) J. Cell Sci. 61, 219-230. morphologically induced during adipocyte differentiation (2). 3. Sargiacomo, M., Sudol, M., Tang, Z.-L. & Lisanti, M. P. (1993) Ligand-bound insulin receptor localizes to adipocyte caveolae J. Cell Biol. 122, 789-807. (27), and insulin stimulation leads to the rapid tyrosine phos- 4. Lisanti, M. P., Scherer, P. E., Vidugiriene, J., Tang, Z.-L., Her- phorylation of caveolin-1 in a time- and dose-dependent manoski-Vosatka, A., Tu, Y.-H., Cook, R. F. & Sargiacomo, M. manner (26). In addition, in response to insulin stimulation, (1994) J. Cell Biol. 126, 111-126. caveolin-1 rapidly associates with an unknown phosphoprotein 5. Chun, M., Liyanage, U., Lisanti, M. P. & Lodish, H. F. (1994) of 30 kDa and Proc. Natl. Acad. Sci. USA 91, 11728-11732. an intracellular pool of caveolin-1 undergoes 6. Chang, W. J., Ying, Y., Rothberg, K., Hooper, N., Turner, A., translocation to the plasma membrane (15, 26). Also, a frac- Gambliel, H., De Gunzburg, J., Mumby, S., Gilman, A. & tion of the insulin-sensitive glucose transporter GLUT4 par- Anderson, R. G. W. (1994) J. Cell Biol. 126, 127-138. titions into caveolae-rich membranes purified from 3T3-LI 7. Shenoy-Scaria, A. M., Dietzen, D. J., Kwong, J., Link, D. C. & adipocytes (15). It is important to determine whether caveo- Lublin, D. M. (1994) J. Cell Biol. 126, 353-363. lin-2 participates in these transmembrane signaling and mem- 8. Robbins, S. M., Quintrell, N. A. & Bishop, M. J. (1995) Mol. Cell. brane trafficking events. Biol. 15, 3507-3515. Is the coexpression of caveolin-1 and caveolin-2 within a 9. Schnitzer, J., McIntosh, D., Dvorak, A. M., Liu, J. & Oh, P. single cell functionally redundant or do they serve distinct but (1995) Science 269, 1435-1439. complementary roles? As caveolin-2 colocalizes with caveo- 10. Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y., Glen- lin-I by cell fractionation and immunolocalization studies, ney, J. R. & Anderson, R. G. W. (1992) Cell 68, 673-682. 11. Glenney, J. R. (1992) FEBS Lett. 314, 45-48. caveolin-2 must also localize to caveolae. However, some 12. Dietzen, D. J., Hastings, W. R. & Lublin, D. M. (1995) J. Biol. evidence suggests that caveolin-1 and caveolin-2 are function- Chem. 270, 6838-6842. ally distinct. Caveolin-1 may function as a GDI for heterotri- 13. Li, S., Okamoto, T., Chun, M., Sargiacomo, M., Casanova, J. E., meric G proteins. A peptide derived from caveolin-1 function- Hansen, S. H., Nishimoto, I. & Lisanti, M. P. (1995) J. Biol. ally interacts with purified trimeric G proteins and suppresses Chem. 270, 15693-15701. basal GTPase activity (13). In contrast, a peptide derived from 14. Scherer, P. E., Tang, Z.-L., Chun, M. C., Sargiacomo, M., Lodish, the corresponding sequence of caveolin-2 functionally acti- H. F. & Lisanti, M. P. (1995) J. Biol. Chem. 270, 16395-16401. vates the GTPase activity of purified trimeric G proteins 15. Scherer, P. E., Lisanti, M. P., Baldini, G., Sargiacomo, M., Cor- without affecting GDP/GTP exchange; caveolin-2 functions as ley-Mastick, C. & Lodish, H. F. (1994) J. Cell Biol. 127, 1233- a GAP. Both the GAP and GDI activities serve to place or 1243. hold G proteins in the inactive GDP-liganded 16. Sargiacomo, M., Scherer, P. E., Tang, Z.-L., Kubler, E., Song, conformation, K. S., Sanders, M. C. & Lisanti, M. P. (1995) Proc. Natl. Acad. suggesting that caveolin-1 and caveolin-2 might act in concert Sci. USA 92, 9407-9411. to sequentially recruit and sequester inactive G proteins within 17. Monier, S., Parton, R. G., Vogel, F., Behlke, J., Henske, A. & caveolae membranes. Specifically, (i) caveolin-2 could function Kurzchalia, T. (1995) Mol. Biol. Cell 6, 911-927. first as a GAP to place activated G proteins in the inactive 18. Baldini, G., Hohl, T., Lin, H. & Lodish, H. F. (1992) Proc. Natl. conformation and recruit them to the caveolae membrane and Acad. Sci. USA 89, 5049-5052. (ii) caveolin-1 could then function as a GDI to hold them in an 19. Kurzchalia, T., Dupree, P., Parton, R. G., Kellner, R., Virta, H., inactive conformation within caveolae membranes. This would Lehnert, M. & Simons, K. (1992) J. Cell Biol. 118, 1003-1014. provide a two-step mechanism for concentrating inactive G 20. Smart, E., Ying, Y.-S., Conrad, P. & Anderson, R. G. W. (1994) proteins within caveolae for presentation to activated G- J. Cell Biol. 127, 1185-1197. protein-coupled receptors. Consistent with this idea, ligand- 21. Lisanti, M. P., Tang, Z.-T., Scherer, P. & Sargiacomo, M. (1995) bound endothelin receptors colocalize with Methods Enzymol. 250, 655-668. caveolin-1 within 22. Sternweis, P. C. & Robishaw, J. D. (1984) J. Biol. Chem. 259, the plasma membrane (5). The mechanism by which G pro- 13806-13813. teins cycle on and off the plasma membrane remains largely 23. Okamoto, T., Katada, T., Murayama, Y., Ui, M., Ogata, E. & unknown (28). In this regard, our current findings could Nishimoto, I. (1990) Cell 62, 709-717. provide a new framework for understanding these molecular 24. Okamoto, T. & Nishimoto, I. (1992) J. Biol. Chem. 267, 8342- signaling events in the context of caveolae. 8346. 25. Tang, Z.-L., Scherer, P. E. & Lisanti, M. P. (1994) Gene 147, We thank Drs. David Baltimore, Monty Krieger, and Anthony J. 299-300. Koleske for critical reviews of the manuscript; Dr. Guilia Baldini for 26. Corley-Mastick, C., Brady, M. J. & Saltiel, A. R. (1995) J. Cell help in Northern blot analysis; Richard F. Cook for microsequence Biol. 129, 1523-1531. analysis; Dr. John R. Glenney for generously donating monoclonal 27. Goldberg, R. I., Smith, R. M. & Jarett, L. (1987) J. Cell. Physiol. antibodies to caveolin-1; and Dr. Tatsuya Haga for purified Go protein. 133, 203-212. This work was supported in part by a grant from the W. M. Keck 28. Wedegaertner, P. B., Wilson, P. T. & Bourne, H. R. (1995) J. Foundation to the Whitehead Fellows program (M.P.L.), a National Biol. Chem. 270, 503-506. Downloaded by guest on September 24, 2021