The Caveolin Scaffold Hypothesis

The scaffold hypothesis is a widely cited early interpretation of the link between caveolin and caveola-associated proteins [84]. This hypothesis proposes that proteins associated in vivo with caveolin can be identified by a "scaffold recognition" sequence that is responsible for their co-assembly, and identifies the protein-protein contact domain. Based on the selectivity of a peptide library, the scaffold sequence motif was identified as -FxxFxxxxF-, FxxxxF xxF- or -fxfxxxxf- where F is an aromatic amino acid (F, W, or Y) and x is any residue. The orientation of the signature aromatic amino acids in these linear motifs varies, depending on whether it forms part of a loop, beta-sheet or alpha-helical sequence within the tertiary structure of the native protein.

In terms of this definition, caveolin itself contains a scaffold sequence or rather, two overlapping sequences (Fig. 5.3A). One of these overlaps a sequence (-K96YW-FYR-) which is implicated in membrane binding ("membrane attachment sequence") [85]. GFP-caveolin peptide lacking this sequence retains about 50% of membrane binding. One scaffold sequence is mainly predicted as helix, the other as beta-sheet. The cav(82-101) domain also includes a "cholesterol recognition amino acid consensus (CRAC-1)" sequence -L/V(x1-4)Y (x1-4)K/R- [86] that partially overlaps the "scaffold" sequences.

Lipid Rafts
Fig. 5.3 (A) Location of predicted scaffold motifs and FC recognition sites of the cav(82-101) domain of human caveolin-1 (from [84,86]. (B) Location of amino acid residues needed for association G1a, eNOS, and FC with caveolin (from [80,82,85,96]).

Does the Scaffold Motif in Signaling Proteins that are Present in Caveolae Represent the Contact Site of these Proteins with Caveolin?

In the eNOS complex with caveolin (which is perhaps the best-studied example), the scaffold sequence is -F347SAAPFSGW-. This was recently identified as part of an internal loop stabilizing the heme prosthetic group [87], and so it is unlikely to be accessible to caveolin [80]. -F656SYVNPQF- in protein kinase C is within a closed conformation of the V5/C3/4 domain [88]. The scaffold sequence in endo-thelin receptor-A (ER-A, -WPFDHNDFGVF-) is part of extracellular loop-1 [89]. Since caveolin lacks an extracellular domain [43], the scaffold motif in ER-A cannot represent a functional protein-protein contact site. In other cases, structural information is inadequate to assign the location of the scaffold motif within the three-dimensional structure of the protein. Overall, however, the experimental data are lacking that, in a native complex of caveolar proteins, the "scaffold sequence" identifies a site that binds caveolin.

Table 5.1 Peptide contributions to free-cholesterol (FC)-bind-ing sites.













Benzodiazepine receptorb



HIV-1 Nefb






a From X-ray crystallography. b From FC or sterol analogue-binding.

a From X-ray crystallography. b From FC or sterol analogue-binding.

Another test of the scaffold hypothesis for signaling proteins can be made. Does the presence or absence of a scaffold sequence predict whether a cell membrane protein binds to caveolin? In Table 5.1, the frequency of scaffold sites was seen to be one per 541 amino acids within the primary sequence of caveola-associated signaling proteins. However, a quite similar incidence (one in 493 residues) was present in lipid-associated proteins not associated with caveolin or caveolae (NPC-1, HMG-CoA reductase, lecithin:cholesterol acyltransferase, acyl CoA cholesterol acyltransferase, clathrin heavy chain) (sequences data from GenBank). This finding argues strongly against the diagnostic significance of the scaffold motif in signaling proteins.

Two rather different questions relate the F,W,Y-rich "scaffold" sequences in caveolin.

• Is the domain of caveolin that includes these sequences the contact site with signaling proteins in caveolae?

• If so, are the aromatic amino acids that define the scaffold sequence necessary for this contact?

There can be little doubt that the central domain of caveolin (cav82-101) is somehow important in both promoting protein-protein and protein-lipid associations in caveolae. Evidence supporting this includes its ability both in vitro [90] and in vivo [78] to compete with caveolin to affect signal protein binding and activity (Table 5.1). But is it the pattern of three aromatic residues that is important?

Inconsistent with the scaffold hypothesis, alanine-scanning mutagenesis showed that an intact scaffold sequence was not required for caveolin-derived pep-tides to bind to G1a [84]. Within the cav(82-101) domain, only residues -F92TVT-were needed for binding activity (Fig. 5.3B). Nor was a complete scaffold sequence required to compete with caveolin in displacing eNOS [80]. In a third case, that of adenyl cyclase [90], a different pattern of amino acids, including elements of the FC recognition site, was needed for caveolin binding.

Though information of this same kind is needed for additional proteins, the data so far argue against the significance of a meaningful "scaffold sequence." Proteins bound to caveolin are no more likely to include a scaffold sequence than other proteins. The scaffold sequences in caveolin appears to be parts of a broader pattern of amino acids within the cav(82-101) domain responsible both for the association of caveolin with signaling proteins, and with FC.

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